| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, June 19,
1995; and in revised form, September 28, 1995) From the
We have shown previously that the active form of avian sarcoma
virus integrase (ASV IN) is a multimer. In this report we investigate
IN multimerization properties by a variety of methods that include size
exclusion chromatography, chemical cross-linking, and protein overlay
assays. We show that removal of the nonconserved C-terminal region of
IN results in a reduced capacity for multimerization, whereas deletion
of the first 38 amino acids has little effect on the oligomeric state.
Binding of a full-length IN fusion protein to various IN fragments
indicates that sequences in both the catalytic core (residues
50-207) and a C-terminal region (residues 201-240)
contribute to IN self-association. We also observe that the isolated
C-terminal fragment (residues 201-286) is capable of
self-association. Finally, a single amino acid substitution in the core
domain (S85G) produces a severe defect in multimerization. We conclude
from these analyses that both the catalytic core and a region in the
nonconserved C terminus are involved in ASV integrase multimerization.
These results enhance our understanding of integrase self-association
determinants and define a major role of the C-terminal region of ASV
integrase in this process.
The integration of viral DNA into the genome of the host cell is
a unique and vital step in the normal life cycle of retroviruses (for
recent reviews, see (1) and (2) ). The retroviral
integrase (IN) (
Figure 1:
Domain structure of retroviral
integrases. The figure shows the relative positions of the major
domains along the linear sequence of retroviral integrases. The scale
of amino acid numbering is indicated at the top (ASV IN
= 286 amino acids; HIV-1 IN = 288 amino acids). The
catalytic core is an evolutionarily conserved region among retroviral
INs and certain transposases; the acidic residues (D,D(35)E) presumed
to bind the divalent cations required for activity have been positioned
to scale. Another conserved motif, the HHCC region, is located in the
N-terminal region. It contains appropriately spaced histidines and
cysteines characteristic of several Zn
Many enzymes that catalyze DNA
recombination require the formation of multimeric protein-DNA
complexes. Detailed structural information is available for some of
these(18, 19, 20, 21, 22) .
However, important questions concerning the stoichiometry of IN protein
and DNA substrates in the nucleoprotein complex that is competent for
integration remain unanswered. In addition, the role of protein
multimerization in substrate binding and catalysis is yet to be clearly
defined. Both biochemical and genetic studies indicate that
multimerization is a functionally important property of retroviral
integrases. ASV IN, purified from viral particles, and bacterially
expressed HIV-1 IN were observed to migrate in glycerol gradients at a
position consistent with a dimer molecular
weight(23, 24) . Gel filtration studies also suggest
that purified HIV-1 IN forms dimers(25) . We have used
sedimentation and kinetic studies to demonstrate that purified ASV IN,
which exhibits a reversible mass action between monomer The work presented here includes
physical analyses of the multimeric state of purified ASV IN and
various IN fragments using size exclusion chromatography (SEC) and
chemical cross-linking techniques. To further delineate the regions of
this enzyme that contribute to self-association, we have also employed
a modification of a protein overlay blot technique which tests directly
for binding between two potential associating proteins. In addition to
demonstrating a role for the conserved catalytic core domain in ASV IN
dimerization, our results uncover an important determinant for
multimerization located in the less conserved C-terminal domain.
The
IN(201-286)st clone, which expressed the IN fragment
201-286, included a streptavidin epitope tag at the C terminus.
It was constructed in three steps as follows. 1) A DNA duplex fragment
encoding a kinase/strep-tag sequence (35) of 15 amino acids
(Arg-Arg-Ala-Ser-Val-Ser-Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly) with
compatible ends was inserted into BamHI/HindIII-digested pSE380(36) . 2) A
polymerase chain reaction fragment encoding IN amino acids
201-286 (using primers with flanking NcoI and BamHI sites) was inserted into the pSE380 strep-tag
derivative. 3) The IN-strep-tag encoding DNA fragment was purified and
ligated to NcoI/HindIII-digested expression vector
pET-20b (Novagen). The polypeptide expressed from this construct
includes a leader sequence (pelB) from the pET-20b vector, which is
intended for periplasmic targeting of overexpressed proteins. However,
the most effective purification of IN(201-286)st was from whole
cell lysates. The apparent molecular weight from SDS gels indicate that
this leader is cleaved by a signal peptidase. The IN sequences of all
clones were confirmed by sequencing, and fusion proteins of expected
molecular weight were synthesized in all cases.
For
labeling purposes, glutathione beads with bound fusion protein were
washed with kinase buffer (20 mM Tris-Cl, pH 7.5, 0.1 M NaCl, 12 mM MgCl
The SEC analyses
presented do not attempt to account for the dissociation kinetics of IN
multimers. The observation of discrete monomer and dimer peaks in some
chromatograms (for example, Fig. 4, traces 4 and 5) indicates that the dissociation rate of dimer to monomer
must be slow relative to the column run time. The intent of the
experiments presented here was to compare the behavior of different
polypeptides and not to establish absolute quantitative constants for
IN self-association. Sedimentation equilibrium analysis is more suited
to the quantitative determination of association constants, and the
results of such studies (in progress) will be reported separately.
Figure 4:
Size exclusion chromatography of
full-length IN and truncated IN proteins. Size exclusion chromatography
was performed as described under ``Materials and Methods.''
The chromatograms display the absorbance at 220 nm as a function of
elution time. The trace numbers noted in the text are found to the left of each chromatogram. For reference, the elution
positions of 2 (of a total of 7) globular molecular mass standards are
indicated with dotted vertical lines. The loading
concentration of each polypeptide was approximately 30 µM. Lightly shaded traces indicate proteins that are competent for
multimerization; hashed traces indicate proteins partially
defective in multimerization, and darkly shaded traces indicate proteins most defective in multimerization. We note that
full-length IN elutes with an apparent molecular mass that can be best
assigned as a dimer (
The
experiment in Fig. 6uses GST-fusion proteins for both probe and
targets. GST has been shown to be a dimer in the active
state(37) . Although we included excess unlabeled, nonfused GST
as competitor, this assay may detect some GST-GST interactions between
probe and targets. The level of probe binding to GST alone is indicated
and should be considered the background for each probe. Note that the
binding of the gIN(1-286) probe to the full-length target is
greater than 20-fold above binding to GST alone, demonstrating an
adequate signal-to-noise ratio under our assay conditions. Experiments
were repeated with several of the constructs using nonfused protein
targets (data not shown), and similar results were obtained.
Figure 6:
Mapping determinants of IN
self-association. The left part of the figure shows the set of
truncated proteins generated to test for probe binding. The dark
bars indicate the portions of IN included in each GST-fusion, and
the corresponding name is listed in the first column of the
table to the right. Dotted lines delineate the catalytic core
and C-terminal regions proposed to be involved in ASV IN
self-association. The variable shading of the C-terminal
self-association region (here and in Fig. 1) indicates the
relative contribution to multimerization conferred by the sequences
within this region. The last two columns of the table present
the normalized data for binding of two different probes, full-length IN
fusion, gIN(1-286), and gIN(201-236), to each target
protein. ND indicates not done.
Figure 2:
Chemical cross-linking of IN subunits. A, titration of the cross-linking reagent DSP. Reactions
containing 7 µM IN (wild type, nonfused) and various
concentrations of DSP were incubated for 30 min at 22 °C. They were
then quenched by addition of a molar excess of glycine and an equal
volume of SDS sample buffer without a reducing agent (except in lane 8 where 2-mercaptoethanol was included). Samples were
analyzed by 12% SDS-PAGE and silver staining. The positions of the
molecular mass markers are noted to the left; positions of IN
multimers are indicated to the right (1-IN =
IN monomer, 2-IN = IN dimer, 4-IN = IN
tetramer). The concentrations of DSP in lanes 2, 3, 4, 5, 6, and 7 were 2
µM, 20 µM, 0.2 mM, 1 mM, 2
mM, and 20 mM, respectively. Lane 8 contained 2 mM DSP. The presence of trace amounts of
dimer in the lane with no cross-linker is due to fortuitous disulfide
bonds formed between native IN monomers that are not reduced, since the
gel sample buffer could not contain reducing reagents with this
cross-linker. Protein in a secondary band (estimated 28 kDa) below
full-length IN monomer (32 kDa) is derived from full-length IN, since
it is recognized in Western blots with monoclonal antibodies directed
against ASV IN (data not shown). Cross-linking of this fragment can
account for some of the broadness of the dimer band seen in this figure
and Fig. 3. B, cross-linking with the reagent EDC.
Reactions containing 10 µM IN, 650 mM NaCl, and
increasing concentrations of EDC were incubated for 30 min at 22
°C. Labeling is as in A. The concentrations of EDC in lanes 1, 2, 3, 4, and 5 were 8 µM, 40 µM, 0.2 mM, 1
mM, and 5 mM, respectively. C, chemical
cross-linking with lower concentrations of IN. Concentrations of IN in lanes 2, 3, 4, and 5 were 7
µM, 3.5 µM, 70 nM, and 350
nM, respectively. The assignment of the band labeled 4-IN (tetramer) is more apparent in lower percentage gels. Reaction
mixtures contained 0.5 M NaCl, 0.2 mM BS
Figure 3:
Comparison of multimerization by
full-length IN and truncated IN proteins. A, comparison of
full-length IN and IN(1-207). Lanes 2-5 contained
3.5 µM full-length IN (wild type), and lanes 6-9 contained 4.8 µM IN(1-207). Concentrations of
BS
Other reagents with different
chemistries (e.g. glutaraldehyde and dimethyl
3,3`-dithiobispropionimidate) were also examined for their ability to
covalently cross-link IN multimers. Multimeric complexes similar in
composition and amount to those observed with DSP and EDC were detected
with these reagents (data not shown), suggesting that a variety of
reactive residues (basic, acidic, and others) must be present at or
near the interface between monomer subunits. Experiments in which
the IN concentration was decreased while the cross-linker concentration
was held constant showed that dimers are present at IN concentrations
as low as 70 nM (Fig. 2C). These data also
reveal a concentration dependence for tetramer formation (Fig. 2C, compare lanes 2 and 3).
These results are consistent with previous estimates of a K
The
cross-linker BS No heterodimerization was observed in mixtures of full-length and
IN(1-207) polypeptides in this assay (data not shown). It is
possible that under these experimental conditions, exchange of monomer
subunits proceeds too slowly for heterodimers to form. However, this
could also reflect the fact that the affinity of an IN(1-207)
subunit for the full-length IN is significantly lower than two
full-length IN monomers for each other.
In the course of site-directed mutagenesis studies, our laboratory
has prepared a number of altered ASV IN proteins that contain single
amino acid substitutions in residues that are highly conserved in
retroviruses and certain other transposable elements(11) . A
number of these proteins (D64E, T66A, F126A, D121E, L163A, H9N, K206A,
R227A) were examined by SEC (data not shown). Only one of these, S85G,
exhibited a significant difference when compared with wild type
protein. As illustrated in Fig. 4(trace 2), S85G
eluted exclusively as a monomer. The results of these SEC analyses
are in general agreement with those of the chemical cross-linking
studies. Both sets of data indicate that the catalytic core domain of
ASV IN can dimerize, but with reduced efficiency compared to the
full-length protein. Addition of the C-terminal region appears to
restore full multimerization capability. We conclude that
self-association determinants are located in both the core and
C-terminal regions of ASV IN. The inability of the S85G mutant to
dimerize suggests that substitutions in this residue alter the
catalytic core structure, or the way in which the core interacts with
the C-terminal domain. Analysis of the crystal structure of ASV
IN(52-207) reveals that the side chain of this residue
participates in a network of hydrogen bonds in a tight turn between two
Figure 5:
Protein-protein association detected by
labeled protein overlay: comparison of full-length IN and
IN(1-207). Nonfused, full-length IN(1-286) and
IN(1-207) were blotted, renatured, and tested for binding to the
labeled probe of full-length IN fused to GST, gIN(1-286), as
described under ``Materials and Methods.'' Bovine serum
albumin (BSA) and other molecular mass standards (MW)
were included as controls. The binding of the labeled probe to some of
these molecular mass proteins is lower than the background level of
binding to the membrane alone. A shows a Coomassie-stained gel
(12.5%) loaded identically to the gel blot in B. The position
of molecular mass markers are noted at the left.
The
C-terminal truncation protein showed reduced binding (Fig. 5B, lane 4), even though equal molar
amounts of this polypeptide were used in the assay (Fig. 5A, compare lanes 3 and 4). The
relative amount of probe bound was quantitated by radioanalytic imaging
and normalized to the amount of protein present on the filter. These
calculations indicated that the C-terminal truncation protein bound to
the probe with 5- to 10-fold lower efficiency than the full-length IN.
Since these results were consistent with those from our physical assays
of IN multimerization, we used this method to screen a series of nested
N-terminal truncated IN proteins. For ease and uniformity of
purification, these truncated proteins were constructed as GST-fusion
proteins (see Fig. 6and ``Materials and Methods'').
The fusion proteins were expressed in E. coli,
affinity-purified, and used as targets in the protein overlay assay.
The relative binding capacity of these proteins was tested with two
probes, full-length gIN(1-286) and gIN(201-236), and
quantitated as described above. Fig. 6provides a map of the IN
deletion proteins tested and a summary of the results of this
quantitation expressed as percent of probe bound to a full-length IN
target protein. Deletion of the N-terminal HHCC domain did not
significantly change the binding with full-length probe (compare the
gIN(1-286) and gIN(60-286) targets), consistent with
results from SEC and cross-linking with the IN(39-286) protein.
Further N-terminal truncation, which removed part
[gIN(120-286)] or all [gIN(156-286)] of
the D,D(35)E region, reduced binding to an intermediate level,
40-60%. Continued truncation did not change binding significantly
until the deletion extended into the C-terminal domain which includes
amino acids downstream of residue 201. After that, binding of the probe
continued to decrease [gIN(223-286)] until the deletion
included amino acid 239 [gIN(240-286)], when it reached
a background level, equivalent to binding to the GST alone control. These results delineate two regions critical to IN self-association.
The first corresponds to the central catalytic D,D(35)E domain, and the
second lies in a C-terminal domain including, but perhaps not limited
to, amino acids 201-240 of ASV IN. Results with targets which are
truncated from the C terminus were consistent with this designation.
The target protein gIN(1-236) bound the full-length probe with
approximately 70% efficiency, whereas the efficiency with
IN(1-207) was only 26%. The association properties of the
C-terminal domain were further investigated by probing the same set of
target proteins with the gIN(201-236) fusion protein. The results
with this probe revealed a pattern distinct from that of the
full-length probe. All of the N-terminal truncation proteins up to and
including gIN(223-286) bound the gIN(201-236) probe with
approximately 70-80% the efficiency of the full-length target (Fig. 6, last column). The C-terminal truncation
IN(1-236) bound this probe as well as or better than the
full-length IN, but no binding above background was observed to the
IN(1-207) target. It is possible that this method could detect
both quaternary interactions between IN monomers and tertiary
interactions that reflect the folding of domains within an IN monomer
polypeptide. However, results with the gIN(201-236) probe, which
showed equivalent binding to all targets that contained residues
201-240, make it unlikely that this reaction is simply mimicking
tertiary interactions. They suggest, instead, that this C-terminal
peptide is capable of specific association with the homologous region
in another target polypeptide.
Figure 7:
Self-association of the C-terminal IN
fragment IN(201-286)st. SEC was performed using the C-terminal
fragment IN(201-286)st as in Fig. 4. The chromatogram
shows two peaks: the first eluted in the void volume of the column, and
the second (labeled IN(201-286)) eluted at a position consistent
with a multimer of the IN(201-286)st fragment. The multimer peak
was confirmed to contain the IN(201-286)st fragment by SDS-PAGE
analysis of the fractions. The 260:280 nm absorbance ratio of the void
peak was consistent with the presence of both nucleic acid and protein
components in this fraction, which could account for the observed
aggregation. The inset shows results from a chemical
cross-linking experiment with the IN(201-286)st fragment and
cross-linker BS
Results obtained with these nonequilibrium methods are
consistent with independent estimates of dissociation constants
determined from sedimentation equilibrium experiments. Whereas
full-length ASV IN has a K Both the
cross-linking and SEC methods record the behavior of the majority of
molecules in the protein preparations, whereas multimerization inferred
from enzymatic complementation (30, 31) or a
transcriptional reporter system (32) could reflect the activity
of a small fraction of protein present. In addition, the latter assays
include DNA substrates which could facilitate the formation of
multimers of IN. We have performed cross-linking experiments in the
presence of various DNA substrates and have failed to detect
enhancement of ASV IN multimerization (data not shown). However, this
could reflect the adverse affects of the high salt conditions required
to keep the protein soluble. We note also that enzymatic
complementation cannot identify regions of IN that contribute to
self-association if they do not include the minimal region necessary
for catalytic activity. The analyses reported here do not require
overlap with catalytic regions and represent the first evidence that
important determinants of self-association reside in the C-terminal
region of a retroviral integrase.
Since multimerization is required
for IN function, inhibitors of self-association may be of potential use
in antiviral therapy. The protein overlay method may be particularly
suited for the identification of peptide inhibitors that interfere with
this property and presumably viral integration. There is precedent for
such an inhibitor strategy with the retroviral
protease(44, 45) .
It is still unclear whether
retroviral integrase functions as a dimer or a tetramer. Formation of a
tetramer might require interactions across two separate protomer
interfaces, one for dimerization and a second for the association of
two dimers into a tetramer. Currently, it is not possible to conclude
that the ASV IN C-terminal domain is involved in either of the
postulated interfaces. However, it is noteworthy that C-terminal
truncated proteins, IN(52-207) and IN(1-207), do not form
tetramers in chemical cross-linking experiments as do full-length IN,
IN(39-286), and IN(201-286) ( Fig. 2and Fig. 6). Whether this is due to the absence of the C-terminal
domain remains to be investigated. The topology of their folding
places the retroviral integrases in a family of nucleases that includes
RNase H, the RuvC resolvase, and the MuA transposase(46) .
Despite sharing a similar fold, and probably similar reaction
chemistries, these diverse nucleases differ in substrate specificity,
multimeric structure, and the requirement for coordination of cleavages
performed. For example, RNase H is known to act as a monomer, whether
as an independent domain or in the context of HIV-1 reverse
transcriptase(47) , and, correspondingly, its function does not
require coordination of multiple cleavages. In contrast, RuvC is known
to act as a dimer and performs two DNA cleavages to resolve a Holliday
junction, but is not involved in joining of DNA strands(48) .
It is apparent from comparison of their crystal structures that the
RuvC dimeric interface (49) differs from that of the IN core
structures. A primary challenge for the future will be to determine how
aspects of protein sequence and structure give rise to the specific
quaternary interactions that allow each of these proteins to perform
their specialized functions. Further study of ASV IN self-association
should help to identify the relevant distinguishing features of
integrase structure.
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29299-29306
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is both necessary and sufficient for the
integration of a linear DNA with viral ends into a target DNA in
vitro(3, 4) . Our knowledge of retroviral
integrase structure and function continues to be refined by
identification of domains of the enzyme that contribute to the various
functions required during integration (Fig. 1). These include DNA binding, catalytic activities, and the multimerization required for the coordinated joining of
processed viral ends to the site of integration in host DNA. Several
investigations have mapped a nonspecific DNA binding domain to
C-terminal portions of both ASV and HIV-1
IN(5, 6, 7, 8, 9) .
Catalytic functions have been localized to the central core domain,
which is resistant to limited proteolysis(10) , and contains a
conserved triad of acidic residues [D,D(35)E] that is
presumed to bind the divalent cations that are required for catalytic
activity(5, 11) . The isolated catalytic domain is
competent to perform a concerted cleavage-ligation activity (12, 13, 14) and contributes to the
recognition of conserved CA residues present at the 3` ends of
retroviral long terminal repeats and other transposable
elements(15) . However, it cannot perform the viral DNA end
processing and joining reactions required for insertion of viral DNA
into target DNA sequences. The crystal structures of the catalytic
cores of both HIV-1 IN and ASV IN have been solved recently (16, 17) .
binding
domains. The amino acid sequence in the C-terminal region (amino acids
beyond 200) is not highly conserved among retroviral INs. It has been
shown to contain determinants for nonspecific DNA binding and, in
addition to the core region, likely plays a role in substrate binding
during catalysis. The location of the two self-association regions for
ASV IN identified in this report are also indicated. The five ASV IN
truncated proteins used in this report are diagrammed below, with the shaded box indicating the portion of IN encoded. The construct
name includes the end points of amino acids contained in each
polypeptide.
dimer
tetramer, must multimerize to perform its catalytic
function(26) . Similar sedimentation analyses have been
performed with HIV-1 IN(27) . The coordinated action of Moloney
murine leukemia virus IN on both ends of viral DNA was demonstrated by
mutagenesis studies and analysis of intermediates produced in
vivo(28) . Our laboratory has recently obtained similar
results with ASV IN in vitro, using DNA substrates that link
two viral DNA end sequences(29) . Multimerization has also been
inferred from the enzymatic complementation of two defective HIV-1 IN
mutants when incubated together(30, 31) . Finally, a
yeast transcriptional reporter system was used to analyze HIV-1 IN
homomeric interactions and to determine a minimal domain required for
self-association(32) .
Plasmids and Cloning
The cloning of nonfused ASV IN and IN fragments has been
described(15) . To produce plasmid DNA clones encoding IN
fragments with the indicated boundaries fused to glutathione S-transferase (GST), polymerase chain reaction was performed
with Vent polymerase (Boehringer) for 25 cycles from the template
pRC23p32(33) , using primers with flanking BamHI and EcoRI sites suitable for insertion in the expression vector,
pGEX2TK (Pharmacia Biotech Inc.). This expression vector encodes a
kinase labeling site at the junction between GST and the cloned insert
of the fusion protein. Polymerase chain reaction fragments were
purified prior to digestion and ligation; cloning and screening
procedures followed standard practice(34) .Protein Expression and Purification
GST Fusion Protein Expression, Purification, and
Labeling
An overnight culture of Escherichia coli MC1061 bearing GST-IN-encoding plasmids was diluted 1:20 in Luria
Broth medium containing ampicillin and grown at 30 °C to an A between 0.8 and 1.0 before expression was
induced by addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 0.2 mM. Growth was continued for 4 h at
30-35 °C prior to harvesting cells by centrifugation for 25
min at 5000 
g at 4 °C. Cell pellets were frozen
and stored at -70 °C. Lysis was accomplished by two passes
through a French press at 20,000 p.s.i. in TNE buffer (20 mM Tris-Cl, pH 8, 1 M NaCl, 1 mM EDTA). After
lysis, Nonidet P-40 was added to a final concentration of 1% (v/v), and
the preparation was incubated with inversion at 4 °C for 10 min,
prior to clearing by centrifugation at 10,000 g for 15
min at 4 °C. The supernatant fraction was collected and then
diluted with an equal volume of 20 mM Tris-Cl, pH 8, 1 mM EDTA, to bring the final NaCl concentration to 0.5 M. A
50% (v/v) slurry of glutathione-agarose (Sigma) was added for affinity
purification by a batch procedure. Fusion proteins were incubated with
the resin for >1 h at 5 °C prior to washing 5 times with NETN
buffer (0.5 M NaCl, 1 mM EDTA, 20 mM
Tris-Cl, pH 8, 0.5% Nonidet P-40). GST-fusion proteins could be stored
at 4 °C immobilized on the glutathione resin (for several weeks)
or, alternatively, eluted from the resin with 20 mM
glutathione (reduced form, Sigma) in elution buffer (50 mM HEPES, pH 8, 0.4 M NaCl, 0.1 M LiCl, 1 mM EDTA, 0.5% (v/v) Brij-35). Eluted fusion protein preparations were
then dialyzed overnight at 5 °C against 50 mM HEPES, pH 8,
0.5 M NaCl, 0.1 mM EDTA, 1% (v/v) thiodiglycol, 40%
(v/v) glycerol for storage at -20 °C or -70 °C.
Throughout the manuscript, GST fusion proteins are denoted with the
prefix of a lowercase g, i.e. gIN(1-286).
, 10 mM
dithiothreitol). Then, 10 to 20 units of protein kinase (catalytic
subunit from bovine heart, Sigma) and 330 µCi of
[-
P]ATP were added, and the mixture was
incubated with agitation for 30-60 min at 4 °C. After
quenching, the glutathione resin was washed 4 times with NETN buffer
prior to elution of the labeled fusion protein as described above.
Strep-tag Protein Expression and
Purification
Induction of expression and cell lysis were
performed essentially as described for GST-fusion proteins above.
Streptavidin-agarose (Pierce) was added to cleared lysates for affinity
purification by a batch procedure. Fusion proteins were exposed to the
resin for longer than 1 h at 5 °C. The resin was then washed 5
times with 20 mM HEPES, pH 8, 0.5 M NaCl, 1% (v/v)
glycerol, 1 mM EDTA. Strep-tag-fusion proteins were eluted
from the resin with 1 mM D-biotin in elution buffer. Eluted
fusion protein was then dialyzed overnight at 5 °C against 50
mM HEPES, pH 8, 0.5 M NaCl, 0.1 mM EDTA, 1%
(v/v) thiodiglycol, 40% (v/v) glycerol.Expression and Purification of Full-length Nonfused IN
and IN Fragments
Full-length ASV IN and various IN fragments
were expressed in Escherichia coli MC1061 and purified as
described previously (15, 26) using an immunoaffinity
column as the final purification step.Chemical Cross-linking
Cross-linking of IN and IN fragments was carried out using a
variety of chemical reagents (Pierce) with buffer and protein
concentrations noted in the figures. Reactions using the reagents DSP
and BS
were performed in 100 mM HEPES buffer, pH
8.0. Reactions with the reagent EDC were performed in 100 mM MES, pH 5.6, and 5 mMN-hydroxysuccinimidyl
ester. Unless otherwise noted, all reactions contained 600 mM NaCl and were quenched with a molar excess of either glycine or
lysine prior to the addition of an equal volume of SDS sample buffer
containing 280 mM 2-mercaptoethanol. The 2-mercaptoethanol was
omitted if the cross-linker contained a disulfide bond for cleavage, i.e. DSP. Samples were subsequently heated at 95 °C for 10
min. Covalently linked multimers were detected by separation in 12% or
15% SDS-polyacrylamide gels and silver staining.Size Exclusion Chromatography of Full-length and
Truncated IN Proteins
Size exclusion chromatography was performed using a Superdex
75HR 10/30 column (Pharmacia) on a Rainin HPLC system with native IN or
nonfused IN fragments. A flow rate of 0.5 ml/min with a mobile phase of
50 mM HEPES, pH 7.5, 0.5 M NaCl, 1% (v/v) glycerol
was used for all experiments. Typically, 25-50 µl of purified
IN polypeptides at a concentration of 30 µM were injected.
Samples were subjected to centrifugation at 10,000 g prior to injection on the column. Absorbance of the column eluate
was monitored at both 280 and 220 nm. Samples from peak fractions were
monitored by SDS-PAGE for the presence of the expected protein species.
The column was calibrated using seven different globular proteins as
molecular weight standards, and the apparent molecular weight of each
sample peak was determined using linear regression of the log of known
molecular weight versus the elution behavior (K or elution time).
57.8 kDa), yet at a time slightly later than
expected for an ideal globular dimer. This behavior is similar to that
observed with HIV-1 IN(25) . The slightly retarded elution time
most likely reflects nonspecific interaction of IN with the column
matrix. This has been observed with other SEC media used for ASV IN
(data not shown) and was observed for the catalytic core of HIV-IN,
where inclusion of CHAPS in the running buffer decreased elution
time(43) .
Protein Overlay Binding Assay
To test the ability of a labeled protein probe to bind to a
defined set of target IN fragments, standard SDS-PAGE was performed on
the target polypeptides prior to electrophoretic transfer to either a
polyvinylidene difluoride or nitrocellulose membrane using a 3-buffer
semidry technique (according to Millipore recommended procedures).
Subsequent steps of blocking, renaturing, and probing were carried out
at 4 °C. For buffers requiring dithiothreitol, this reagent was
added just prior to use. The transfer membrane was blocked in binding
buffer (25 mM HEPES-KOH, pH 7.7, 25 mM NaCl, 5
mM MgCl, 1 mM dithiothreitol) containing
5% (w/v) powdered milk and 0.05% (v/v) Nonidet P-40 (blocking solution)
for at least 1 h (typically overnight). Target polypeptides were
denatured on the membrane by two successive incubations in binding
buffer containing 6 M guanidine HCl for 10 min. Slow
renaturation was accomplished by 6 successive dilutions with an equal
volume of binding buffer, each with a 10-15-min incubation. After
two washes with binding buffer, the membrane was treated once more with
blocking solution for 1 h and for 30 min in blocking solution with only
1% powdered milk. A molar excess of labeled probe protein was diluted
into probe buffer (20 mM HEPES, pH 7.7, 75 mM KCl,
0.1 mM EDTA, 2.5 mM MgCl, 1% powdered
milk, 0.05% Nonidet P-40, 1 mM dithiothreitol). This probe
mixture also included at least a 2-fold molar excess of unlabeled GST
protein to block nonspecific binding to the GST-fusion targets. The
probe was incubated with the renatured blot for greater than 6 h,
followed by three successive 10-min washes in probe buffer alone. The
membrane was then dried and the radioactivity quantitated on a Fuji
BAS1000 phosphoimaging system. The amount of radioactive probe bound to
target bands of interest was normalized to the quantity of the target
polypeptide loaded on the gel, as assessed by densitometric
quantitation of an identically loaded Coomassie-stained gel.
Integrase Multimers Revealed by Chemical
Cross-linking
Chemical cross-linking has been employed
successfully to examine the protein-protein associations of many
multisubunit enzymes. We have used a variety of chemical cross-linking
reagents to identify the multimeric forms of ASV IN that exist in
solution. As shown in Fig. 2A, titration of the
cross-linker DSP revealed dose-dependent formation of covalently linked
forms of IN (lanes 2-6). As the concentration of
cross-linker was increased, the amount of dimers, tetramers, and higher
order multimers increased while the amount of free monomer decreased.
The dominant multimer observed was a dimer. Optimal concentrations of
DSP were between 1 and 2 mM; higher concentrations of the
cross-linker produced aggregates which failed to enter the gel (Fig. 2A, lane 7). The DSP cross-linker
contains a disulfide bond which can be reduced, thereby breaking the
covalent link between subunits. As expected, treatment with
-mercaptoethanol led to the disruption of cross-linked multimers (Fig. 2A, compare lanes 6 and 8). Fig. 2B shows results of treatment with increasing
concentrations of the ``zero-length'' cross-linker EDC. Due
to the short length of the covalent bond formed (the length of a
typical amide bond), detection of cross-linked multimers using this
reagent (lanes 2-5) provided strong evidence that the
subunit association is structurally
significant(38, 39) .
and were incubated for 15 min at 22 °C. No cross-linker was
added in lane 1. Labeling is as in A.
were 20 µM in lanes 3 and 7, 0.2 mM in lanes 4 and 8, and 2
mM in lanes 5 and 9. Incubations were for 15
min at 22 °C. Labeling is as described in Fig. 2. B, comparison of truncated IN proteins with full-length IN for
formation of covalent multimers using BS. Reactions and
labeling of the gel are as described in A. Protein
concentration in each case is 5 µM, and the absence and
presence of cross-linker is indicated above each lane. In the absence
of cross-linker, a minor amount of nonreduced dimer for
IN(39-286) and IN(1-207) persists despite the inclusion of
reducing agent in the loading buffer.
(monomer-dimer) in the 1-5 µM range(26) .Cross-linking Analysis of Truncated Derivatives of
IN
We have previously described a series of N- and C-terminal
deletions in ASV IN (15) . Two of these, IN(1-207) and
IN(52-207), were shown to have lost normal processing and joining
activities, but retained endonuclease and cleavage-ligation activity
with unimolecular substrates that represent an integration
intermediate(15) . Another truncated protein, IN(39-286),
which lacks amino acids 5-38 from the N-terminal region, retains
both processing and joining activities. () was used to determine the ability of these
three truncated forms of IN to form covalently linked dimers in
solution (Fig. 3). The results showed significantly less
cross-linked dimer with IN(1-207) (Fig. 3A, lanes 7-9), compared to full-length IN (Fig. 3A, lanes 3-5). At a concentration
of 2 mM BS in which all full-length IN was
covalently linked in multimeric forms, most of IN(1-207) remained
monomeric. We conclude that IN(1-207) is deficient in
multimerization. Analysis of IN(52-207), which also lacks amino
acids from the N terminus, showed a similar deficiency (Fig. 3B, compare lane 2 with lane
8). In contrast, deletion of residues 5-38 from the
N-terminal segment alone did not cause reduction in multimerization (Fig. 3B, compare lane 2 and lane 4). Multimerization Detected by Size Exclusion
Chromatography
The oligomeric composition of IN and IN truncated
proteins in solution was also investigated by size exclusion
chromatography. In these experiments, the properties of full-length and
truncated proteins were compared at similar molar concentrations. Under
the conditions described for Fig. 4, full-length IN eluted at a
position consistent with a dimer molecular size (trace 1). The
peak was asymmetric, with a shoulder at the position of the monomer. At
lower loading concentrations of IN (not shown), a greater percentage
eluted in the monomer shoulder, as expected for a
concentration-dependent monomer-dimer equilibrium. No dimer was
detected with IN(52-207) under the same conditions; this
polypeptide comprises the catalytic core of ASV IN (trace 6).
However, much higher loading concentrations of IN(52-207) did
reveal a minor peak consistent with a dimer molecular size (data not
shown). An additional 13 amino acids at the N terminus of the catalytic
core, IN(39-207), enhanced dimerization to an intermediate level (trace 5). IN(1-207) eluted with a profile similar to
that observed for IN(39-207). Most of the IN(1-207) protein
eluted as expected for a monomer (apparent molecular mass 23 kDa),
with a smaller peak containing 15-20% of the eluting protein at
the position of a dimer (trace 4). In contrast to C-terminal
truncations, the N-terminal deletion protein IN(39-286) exhibited
an elution pattern like that of the full-length IN (trace 3).
-strands (17) .Localization of Self-associating IN Domains
In
order to map the self-association domains of ASV IN and to gain a
better understanding of their relationship to one another, we used
deletion mutagenesis coupled with a protein overlay technique. This
technique (40, 41) employs a labeled protein to probe
a Western blot of target proteins that are first denatured and then
renatured. We modified this procedure to investigate the
self-association potential of individual regions of ASV IN. For the
first experiment, a labeled GST-IN fusion protein, gIN(1-286),
was the probe for a series of renatured targets which included
full-length IN, IN(1-207), and other non-integrase protein
controls. The data showed efficient binding of the probe to full-length
IN (Fig. 5B, lane 3) and no detectable binding
to the bovine serum albumin and molecular mass standards (Fig. 5B, lanes 1 and 2). Probe
binding to full-length IN could be competed by incubation of the blot
with unlabeled, full-length IN fusion, but not with the GST portion of
the fusion protein alone (data not shown). Therefore, we conclude that
the binding reaction is specific for IN-derived polypeptides.
An IN(201-286) Fragment Can
Self-associate
As a final test of the self-associating
capability of the C-terminal domain, we expressed and partially
purified IN(201-286)st as a strep-tagged protein (35) and
analyzed this polypeptide by SEC and chemical cross-linking (Fig. 7). Under the SEC conditions used, a monomer of this
fragment is expected to elute at 24.5 to 25 min. The results showed
that this C-terminal fragment eluted at 20.5 min, consistent with a
formation of a multimer (trimer or tetramer). Material in the void
volume contained other protein and nucleic acid contaminants, whereas
the multimer peak contained the bulk of the IN(201-286)st
fragment (confirmed by SDS-PAGE analysis of fractions, data not shown).
This C-terminal fragment was also tested in chemical cross-linking
experiments, performed as in Fig. 3B. These results
showed that the C-terminal polypeptide is able to form dimers, trimers,
and tetramers in solution ( Fig. 7inset at top). The
self-association properties of this fragment are not as limited as
those observed with other fragments analyzed. It could be that this
isolated C-terminal fragment is less sterically restrained, allowing it
to form multimers (e.g. trimers) not found with other larger
fragments. We conclude that the C-terminal fragment, IN(201-286),
can self-associate as an independently expressed polypeptide.
under the conditions described in Fig. 3B. The positions of covalently linked dimers,
trimers, and tetramers are noted.
Multimerization Detected by Cross-linking and
SEC
We have investigated the self-association properties of ASV
IN and IN fragments using a variety of methods, each of which offers
distinct advantages and limitations. Chemical cross-linking is the
least stringent of the tests for multimerization because protein
concentration can be controlled to favor association. SEC is the most
stringent assay presented here because dissociative forces due to
dilution are prominent during migration of multimeric proteins through
a column, and the detection of multimers depends on the rate of
dissociation relative to column run times(42) . Accordingly,
chemical cross-linking of the ASV IN catalytic core
[IN(52-207)] clearly reveals dimer formation (Fig. 3), whereas in SEC, this fragment runs predominantly as a
monomer. (monomer-dimer) of
1-5 µM(26) , ASV IN(52-207) has a K (monomer-dimer) in excess of 500
µM. ()The HIV-1 catalytic core (residues
50-212) has been reported to have a stronger association than
that of the analogous ASV fragment, and dimers have been observed with
chemical cross-linking, SEC, and sedimentation analysis of the HIV-1 IN
core fragment(27, 30, 43) .Multimerization Detected by Protein Overlay
The
protein overlay technique provides an intermediate level of stringency
for detection of protein-protein associations. It differs from the
first two solution methods in several ways: protein probes are tested
for binding to immobilized renatured targets, binding conditions are
easily manipulated, and the specificity and sensitivity are high. This
method offers an opportunity to investigate binding to partially
purified target proteins and permits rapid screening of many potential
partners for protein-protein associations. However, unlike the first
two methods, it does not allow direct determination of the multimeric
state and stoichiometry of the interacting partners. From these overlay
experiments, we identified both the catalytic core and C-terminal
domains as contributing to multimerization. We also observed that the
C-terminal domain can specifically associate with itself. This latter
property was confirmed by SEC and chemical cross-linking experiments
with an isolated C-terminal fragment. Considered together, results from
our analyses indicate that the two self-association domains of ASV IN
can act independently, predominantly through interactions between
homologous domains in each monomer. However, our data do not rule out
cooperativity of the two self-association domains in the native
full-length protein, nor can we exclude the possibility of other
interactions not identified by these methods that could also contribute
to the stability of a multimer.Relationship between IN Structure, Multimerization, and
Function
Several aspects of the domain structure of IN proteins
revealed in these studies are consistent with recent information
obtained from x-ray crystallographic analysis of the HIV-1
IN(50-212) (16) and ASV IN(52-207) (17) catalytic cores. In both analyses, an extensive interface
between two monomers in the unit cell was observed, with a large
solvent-excluded surface area. The presence of an extensive dimer
interface in both structures is consistent with the contention that the
catalytic core of one monomer interacts with the core of another.
Despite the similar size of the two core domains, the ASV IN structure
lacks the sixth C-terminal 
helix observed in HIV-1. In the HIV-1
dimer, this helix from one monomer extends out from the structure and
interacts across the dimer interface with the analogous helix from the
second monomer. It is possible that the added stability conferred by
this interaction accounts for the tighter association of the HIV-1 core
dimer relative to the ASV core noted above. The topology of both dimers
suggests that the C-terminal domains of the full-length proteins would
be in close proximity and available for interaction with each other
across a multimeric interface.
),
bis(sulfosuccinimidyl)suberate; MES,
2-(N-morpholino)ethanesulfonic acid; SEC, size exclusion
chromatography; HIV-1, human immunodeficiency virus type 1; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.))
We are grateful to Richard Katz, Barbara
Müller, George Kukolj, and Tom Laue for
encouragement and helpful advice during the course of this work. We are
also indebted to George Merkel for purification of several of the
nonfused proteins used in this work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Ebina, A. G. Chatterjee, R. L. Judson, and H. L. Levin The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein J. Biol. Chem., June 6, 2008; 283(23): 15965 - 15974. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. K. Pandey, S. Bera, J. Zahm, A. Vora, K. Stillmock, D. Hazuda, and D. P. Grandgenett Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate J. Virol., November 15, 2007; 81(22): 12189 - 12199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Lu, H. Z. Ghory, and A. Engelman Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase J. Virol., August 15, 2005; 79(16): 10356 - 10368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Hehl, P. Joshi, G. V. Kalpana, and V. R. Prasad Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins J. Virol., May 15, 2004; 78(10): 5056 - 5067. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Yung, M. Sorin, E.-J. Wang, S. Perumal, D. Ott, and G. V. Kalpana Specificity of Interaction of INI1/hSNF5 with Retroviral Integrases and Its Functional Significance J. Virol., March 1, 2004; 78(5): 2222 - 2231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Appa, C.-G. Shin, P. Lee, and S. A. Chow Role of the Nonspecific DNA-binding Region and alpha Helices within the Core Domain of Retroviral Integrase in Selecting Target DNA Sites for Integration J. Biol. Chem., November 30, 2001; 276(49): 45848 - 45855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Hindmarsh and J. Leis Retroviral DNA Integration Microbiol. Mol. Biol. Rev., December 1, 1999; 63(4): 836 - 843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Petit, O. Schwartz, and F. Mammano Oligomerization within Virions and Subcellular Localization of Human Immunodeficiency Virus Type 1 Integrase J. Virol., June 1, 1999; 73(6): 5079 - 5088. [Abstract] [Full Text]
|
|
|
|
|
|
P. Hindmarsh, T. Ridky, R. Reeves, M. Andrake, A. M. Skalka, and J. Leis HMG Protein Family Members Stimulate Human Immunodeficiency Virus Type 1 and Avian Sarcoma Virus Concerted DNA Integration In Vitro J. Virol., April 1, 1999; 73(4): 2994 - 3003. [Abstract] [Full Text]
|
|
|
|
|
|
S. J. S. Steele and H. L. Levin A Map of Interactions between the Proteins of a Retrotransposon J. Virol., November 1, 1998; 72(11): 9318 - 9322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. P. Lutzke and R. H. A. Plasterk Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding J. Virol., June 1, 1998; 72(6): 4841 - 4848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Gerton, S. Ohgi, M. Olsen, J. DeRisi, and P. O. Brown Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions J. Virol., June 1, 1998; 72(6): 5046 - 5055. [Abstract] [Full Text] [PDF]
|
|
|
