Thermodynamic Dissection of a Low Affinity Protein-Protein Interface Involved in Human Immunodeficiency Virus Assembly*
Marta del Álamo,
José Luis Neira
and
Mauricio G. Mateu ¶
From the
Centro de Biología Molecular "Severo Ochoa"
(CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid
and
Centro de Biología Molecular y
Celular, Universidad Miguel Hernández, Av. Ferrocarril s/n, 03206
Elche, Alicante, Spain
Received for publication, April 29, 2003
, and in revised form, May 16, 2003.
 |
ABSTRACT
|
---|
Homo-dimerization of the capsid protein CA of human immunodeficiency virus
through its C-terminal domain constitutes an early crucial step in the virion
assembly pathway and a potential target for antiviral inhibitors. We have
truncated to alanine the 20 amino acid side chains per monomer that
participate in intersubunit contacts at the CA dimer interface and analyzed
their individual energetic contribution to protein association and stability.
About half of the side chains in the contact epitope are critically involved
in the energetic epitope as their truncation essentially prevented
dimerization. However, dimerization affinity is kept low partly because of the
presence of interfacial side chains whose individual truncation improves
affinity by 220-fold. Many side chains at the interface are
energetically important also for the folding of a monomeric intermediate and
for its conformational rearrangement during dimerization. The thermodynamic
description of this low affinity interface (dissociation constant of
10
µM) was compared with those obtained for the other
protein-protein interfaces, nearly all of them of much higher affinity, that
have been systematically analyzed by mutation. The results reveal differences
that may have been evolutionary selected and that may be exploited for the
design of an effective interfacial inhibitor of human immunodeficiency virus
assembly.
 |
INTRODUCTION
|
---|
Assembly and stability of the complex oligomeric proteins that constitute
the capsids of viruses are mediated by multiple noncovalent interactions
between many protein subunits. An attractive antiviral approach involves the
use of compounds able to interfere with critical intersubunit interactions in
viral oligomers. Potential inhibitors include small molecules and also larger
"interfacial inhibitors" that mimic a part of the interacting
epitope
(14).
Rational design of antiviral agents aimed at inhibiting assembly or
facilitating dissociation of a virus capsid may require not only a detailed
structural knowledge of capsid subunit interfaces but also a thermodynamic
understanding of the individual energetic contribution of interfacial residues
and interactions to subunit association. This is particularly important if the
inhibitor must met the conflicting requirements of a minimum size with maximum
affinity and specificity.
Alanine scanning mutagenesis
(5) provides an excellent
strategy to experimentally determine the energetic contributions of
interfacial side chains to protein-protein interaction. Mutation to Ala of
residues other than Gly or Pro eliminates the targeted side chain beyond
C
and disrupts any interaction that involves that side chain, without
introducing new interactions and with the lowest probability of altering the
conformation of the polypeptide backbone
(5,
6). A few groups have applied
this approach for a systematic thermodynamic dissection of interfaces in
dimeric heterocomplexes
(716)
or small homo-oligomers
(1721).
These studies have substantially advanced the molecular understanding of
protein-protein recognition. In many of the interfaces analyzed, a few
centrally located residues contribute most of the binding energy and are
surrounded by energetically less important contacts
(22); in a few cases, however,
the energy of interaction appears to be distributed across most of the
interface (15,
21).
The structural complexity of viral capsids has generally prevented the
application of quantitative experimental thermodynamic approaches based on the
use of mutants to dissect the energetics of capsid subunit association during
assembly. Fortunately, assembly of some capsids, including that of human
immunodeficiency virus
(HIV),1 involve a
number of discrete oligomerization interfaces between protein domains that may
be amenable to quantitative thermodynamic analyses in isolation. Dimerization
of the capsid protein (CA) of HIV-1 through its C-terminal domain (CA-C) is a
major driving force in virus morphogenesis and budding from the cell
(2326).
The three-dimensional structures of CA from HIV-1 and other retroviruses are
known
(2734).
Image reconstruction by cryo-electron microscopy
(35) and measurements of amide
hydrogen exchange (36) of
HIV-1 capsids formed in vitro have provided direct evidence for the
relevance in the assembled capsid of the homotypic CA-C interactions
determined by x-ray crystallography of the soluble CA-C dimer
(29,
30). Each subunit in the HIV-1
CA-C dimer (CA amino acid residues 147231) is composed of a short
310 helix followed by an extended strand and four
-helices
connected by short loops. The dimerization interface is essentially formed by
the parallel packing of helix 2 from each subunit
(Fig. 1A)
(29,30).
The isolated CA-C domain is not only correctly folded but also dimerizes with
the same affinity as the full-length protein
(29), which has validated its
thermodynamic study. Folding and dimerization of CA-C of HIV-1 is a
three-state reaction in which the polypeptide folds to yield a monomeric
intermediate of low stability, and the intermediate dimerizes in a process
that also involves a tertiary conformational reorganization of each monomeric
subunit (Fig. 1B)
(37).
We have chosen the CA-C dimerization interface to undertake the first
experimental thermodynamic dissection of a protein-protein interface
critically involved in assembly of a virus capsid. The use of different
equilibrium techniques allowed us to perform separate calculations on the
specific free energy contributions of each interfacial side chain to each of
the different processes observed during folding/association of CA-C
(37). The implications for HIV
capsid assembly, the design of interfacial antiviral inhibitors, and
protein-protein recognition are discussed.
 |
EXPERIMENTAL PROCEDURES
|
---|
Mutagenesis and Protein PurificationSite-directed
mutagenesis was performed using the QuikChange kit (Stratagene) on recombinant
plasmid pET21b(+) containing the segment corresponding to CA-C of HIV-1
(strain BH10; CA residues 146231). The mutations introduced were
confirmed by sequencing the entire CA-C coding region. The mutant proteins
were expressed in Escherichia coli BL21(DE3) and purified as
described for wild-type CA-C
(37). The proteins were stored
and analyzed in 25 mM sodium phosphate buffer, pH 7.3, unless
specified otherwise. Purified CA-C mutants were run in overloaded SDS-PAGE
gels and found free of contaminants. Their concentration was determined by
UV-spectrophotometry as described
(37).
Determination of Affinity Constants by Analytical Gel Filtration
ChromatographyAssociation constants for the homodimeric mutants
were determined by gel filtration using frontal (large zone) elution
(38). 15-ml aliquots of CA-C
samples with a total protein (monomer) concentration
(Ct) usually ranging from 0.5 to 60
µM were serially applied to a recalibrated Superdex 75 HR FPLC
column (Amersham Biosciences), which had been thoroughly equilibrated with the
appropriate buffer (25 mM sodium phosphate, pH 7.3, unless
specified otherwise) and kept at 23 °C. Samples were eluted at 1 ml/min
and continuously monitored with an on-line UV detector at a wavelength of 280
nm. The elution volume Ve at each
Ct was determined as the midpoint of the
ascending frontal profile in the chromatogram. The weight average partition
coefficient (39) at each
Ct (
) was calculated
using the expression,
 | (Eq. 1) |
where Vt is the total column volume and
V0 is the void volume, which were determined as described
(18). The experimental
w values obtained at different
Ct were fitted to the equation
(39),
 | (Eq. 2) |
where
m and
d are the
partition coefficient of monomer and dimer, respectively, and
Ka is the equilibrium association constant. The
free energy of association
Ga was
calculated by using the equation,
 | (Eq. 3) |
where R is the gas constant and T the absolute temperature.
Zonal (small zone) elution experiments were carried out in the same way,
except that 200-µl aliquots were applied to the column and that the
Ve was defined by the peak position in the
chromatogram. Gel filtration analysis using small zone elution does not
normally provide good Ka values
(38), and it was not used for
most CA-C mutants. However, we found for nonmutated CA-C (and for another,
unrelated dimeric protein) that in our conditions, the
Ka obtained using small zone gel filtration was
coincident within error with that obtained using frontal elution gel
filtration or other standard techniques (see below). Thus, for those CA-C
mutants that we found essentially in the monomeric form at all but the highest
protein concentrations tested, a rough estimation of the
Ka was obtained as follows: the frontal elution
data obtained at µM concentrations were complemented by small
zone elution data obtained at much higher (around 1 mM)
concentrations (that were prohibitive for large zone experiments), and the
combined data were fitted using Equation
2. Because of the extremely low affinity of these particular
mutants, to achieve meaningful fitting values for
m
and
d, we had to assume that at extremely high
(unreachable) concentrations (around 1 M), the protein would be
essentially in the dimeric form and that
d did not
significantly change upon mutation. Use of these reasonable theoretical
assumptions allowed a good fitting of the experimental data for the very low
affinity mutants.
Fluorescence and Circular Dichroism (CD) Spectroscopy and
Dissociation/Unfolding Equilibrium Data
AnalysesDissociation/denaturation of CA-C mutants by guanidinium
chloride (GdmHCl) were spectroscopically probed as described for wild-type
CA-C (37) using a Varian Cary
Eclipse luminescence spectrophotometer and a Jasco 500 spectropolarimeter
equipped with temperature control units. Total protein (monomer) concentration
was 200 µM unless indicated otherwise. Variations in
fluorescence probed dissociation of the dimer into a monomeric form, and the
data were fitted to a dimer-to-monomer two-state transition. Variations in CD
probed the unfolding of the monomeric intermediate, and the data were fitted
to a unimolecular two-state transition. The equations used in Ref.
37 and the program
Kaleidagraph (Abelbeck Software) were used.
Molecular Modeling and Computer AnalysesThe
three-dimensional structure of CA-C
(29,
30) was inspected using
personal computers, a Silicon Graphics work station, and the programs
InsightII (Biosym Technologies) and RasMol
(40). The program Whatif
(41) was used for analyses of
the number, nature, and structural parameters of the interdimer contacts,
solvent accessibility calculations, and modeling of the mutations
introduced.
 |
RESULTS
|
---|
Energetic Contribution of Interfacial Residues to Dimerization of CA-C
MonomersAnalysis of intersubunit interactions in the
crystallographic structure of the CA-C dimer revealed that 22 side chains per
monomer are involved in the contact epitope
(29,
30). We have individually
truncated all of these side chains (except Ala-177 and Pro-207) to alanine.
Dissociation of nonmutated CA-C and each mutant by simple dilution was
quantitated in analytical gel filtration experiments
(Fig. 2 and
Table I). This allowed
determination of the association constant Ka and
the free energy difference
Ga between
dimer and monomer, and thus, the contribution of quaternary interactions to
the dissociation process (37,
42). The difference between
Ga for nonmutated CA-C and
Ga for each mutant
(
Ga) was taken as a measure of the
relative energetic contribution of the truncated side chain (beyond C
)
(Fig. 3, black bars,
and Fig. 4A).

View larger version (16K):
[in this window]
[in a new window]
|
FIG. 2. Dissociation curves obtained by analytical gel filtration using frontal
elution for nonmutated CA-C and some mutants. A, in phosphate
buffer, pH 7.3; B, in phosphate buffer, pH 7.3, with 150
mM NaCl. The curves were fitted to
Equation 2 under
"Experimental Procedures." Circles, parental CA-C;
triangles, mutant T148A; crosses, D152A; squares,
T188A; diamonds, L172A; inverted triangles, Q192A. For very
low affinity mutants T188A and L172A, the fitting needed one additional data
point obtained at a very high protein concentration using zonal elution.
|
|

View larger version (49K):
[in this window]
[in a new window]
|
FIG. 4. Energetic dissection of the CA-C dimerization interface. A,
effect on CA-C association of side-chain truncations. A space-filling model of
one of the monomeric subunits in the CA-C dimer is represented. The
interfacial residues are color-coded according to the effect of
side-chain truncations on Ka and
 Ga. Red, truncation led to
monomeric CA-C at all but the highest concentrations (estimated
 Ga > 6 kcal/mol);
orange, truncation had a substantial but not dramatic effect on
affinity (Thr-188,  Ga = 1.1
kcal/mol); green, truncation did not significantly affect the
affinity ( Ga < ±0.3
kcal/mol) or had only a small negative effect (Lys-199,
 Ga = 0.5 kcal/mol);
violet, truncation increased the affinity
( Ga = 0.4 to 1.8
kcal/mol). B, location of residues Arg-154 and Lys-199
(cyan), Gln-192 (yellow), Ser-178 (green), and
Glu-180 (red), represented as space-filling models, on the CA-C dimer
structure (ribbon model). Residues are labeled a or
b to distinguish between subunits. Individual truncation of the side
chains of Gln-192, Ser-178, or Glu-180 increased dimerization affinity (see
"Results").
|
|
Only five (about one-fourth) of the side chains in the contact epitope
could be truncated with no or very minor effects on dimerization (absolute
values of 
Ga below 0.3 kcal/mol;
Table I and
Fig. 3). These included Thr-148
and Val-191, which participated in a few intersubunit hydrophobic contacts;
Asn-193, which was involved in two reciprocal, contiguous intersubunit
hydrogen bonds with Gln-192; and Asp-152 and Lys-203 which participated in no
intersubunit contacts except two reciprocal, solvent-exposed salt bridges.
These interactions may thus contribute to the free energy of association in a
negligible way. However, an alignment of more than 240 CA sequences from
HIV-1, HIV-2 or simian immunodeficiency virus (SIV) from the HIV sequence data
base
(hiv-web.lanl.gov),
showed that residue 152 was invariably Asp (Glu in one variant) and that
residue 203 was either Lys or Arg. Conservation of charge at these positions
could be explained because the two 152203 pairs were located at
opposite sides of the interface rim and may favor, through electrostatic
steering (43), the correct
orientation of the monomers during association.
Two of the side chains at the interface (Thr-188 and Lys-199) had a
significant contribution to dimerization
(
Ga = 1.1 or 0.5 kcal/mol,
respectively; Fig. 3). In the
dimer, Thr-188 participated in two intersubunit hydrophobic contacts, whereas
Lys-199 was substantially exposed to solvent. Remarkably, as many as nine
interfacial side chains (nearly one-half of the contact residues) were most
critically involved in the energetic epitope, as their individual truncation
led to monomeric CA-C even at high protein concentrations
(Table I). We were able to
estimate the extremely low association constants for the essentially monomeric
mutants (see "Experimental Procedures" and
Fig. 3). The most critical side
chain was Trp-184 (estimated 
Ga
> 10 kcal/mol). Mutant W184A (and also M185A) had been already found
monomeric (29). In the dimer,
Trp-184 from one monomer is essentially buried within a cavity formed by
residues from the opposite monomer and makes multiple (nine) intersubunit
hydrophobic contacts (30).
Most of the remaining eight side chains that also led to dramatic losses in
the free energy of association (estimated

Ga = 68 kcal/mol, depending
on the mutation) are also nonpolar (Ile-150, Leu-151, Leu-172, Val-181,
Met-185, and Leu-189) and deeply buried in the dimer interface, where they
make between one and seven intersubunit C-C contacts. Together with Trp-184,
they delineate a large central area within the contact epitope
(Fig. 4A). Two charged
residues (Arg-154 and Glu-175) were also critical for dimerization. Neither is
involved in intersubunit hydrogen bonds or salt bridges, but both are
partially buried in the dimer (compare Ref.
30 and
Table I). The energetically
critical residues at the interface were all, except for Leu-150 and Arg-154,
almost absolutely conserved in HIV and SIV. Leu-150 was either completely
conserved (in HIV-1) or replaced only by the chemically similar residues Val
(in most HIV-2 viruses) or Ile (in all SIV variants). Arg-154 was found
replaced by Lys in many variants, but this substitution preserved the positive
charge (see below). It must be noted here that in sequence analyses of CA
residue conservation, we occasionally found very rare variants that did not
fit the observed trends. Among other possibilities, such exceptional mutations
may derive from nonfunctional proviruses
(44). These exceptions were
not found in an alignment of a subset of CA sequences that were clearly
derived from infectious virions.
Finally, it was found that truncation to Ala of any of the four remaining
interfacial side chains (Ser-178, Glu-180, Glu-187, and Gln-192, one-fifth of
the residues in the contact epitope) reproducibly increased the
affinity between 2- and 20-fold (Table
I). Very similar results were obtained at two different ionic
strengths (in the presence or absence of 150 mM NaCl; compare
Fig. 2 and
Table I). Truncation of Gln-192
increased the association constant by more than one order of magnitude. The
Gln-192 side chains from the two subunits contact each other and are located
just underneath a cluster of four positive charges (the guanido group of
Arg-154 and amino group of Lys-199 from the two monomers), located at the
interface rim. These four charged groups are close to each other
(Fig. 4B, 4.4 Å
between N
of Arg-154 and N
of Lys-199) and may be involved in
medium range electrostatic repulsions; truncation of both Gln-192 side chains
may create a large cavity that could favor a local conformational
rearrangement and allow separation of those four positive charges, thus
diminishing their mutual electrostatic repulsion. In all but one of the 240 CA
variants analyzed, Arg-154 and Lys-199 were either preserved or mutated to Lys
or Arg, respectively, and Gln-192 was almost absolutely conserved. Other
effects may, however, complicate the situation, as truncation of Arg-154
prevented dimerization and that of Lys-199 led to a small decrease in
Ka (see above). Ser-178 and Glu-180, whose
individual truncation also improved affinity, are involved in reciprocal
intersubunit hydrogen bonds, and the two pairs of side chains form a cluster
at the interface rim (Fig.
4B). The carboxyl group of Glu-180 also binds a water
molecule that is in turn hydrogen-bonded to the main chain oxygen of Gln-176
from the opposite subunit. As discussed for Arg-154/Lys-199, the two Glu-180
carboxylates are spatially very close in the dimer structure
(Fig. 4B, 3.7 Å
between both O
) and may electrostatically repel each other. This could
explain the higher affinity of the E180A mutant. Truncation of the two serine
side chains in S178A may allow some separation of the Glu-180 carboxylates,
thus decreasing their mutual repulsion and leading to an increase in affinity.
Ser-178 and Glu-180 were not completely conserved. Yet, in practically every
HIV or SIV variant analyzed, including several hundred HIV-1 variants from the
European Molecular Biology Laboratory (EMBL) data base, a negatively charged
side chain was preserved either at position 180 (Glu or Asp) or at position
178 (Asp), and a small size, neutral side chain was found at the other
position (in most cases, Ser or Thr at 178 or Ala at 180). This and the
relative orientations of these side chains
(Fig. 4B) suggest that
a repulsive intersubunit interaction between a pair of negatively charged
residues in this particular spot of the CA-C dimer interface may be preserved
in essentially all HIV and SIV variants. Further experiments (underway) are
needed to validate the above hypotheses on the negative effects and biological
conservation of some interfacial residues on CA-C dimerization (see
"Discussion").
Energetic Contribution of the Interfacial Side Chains to the
Conformational Reorganization of the CA-C Monomer during
DimerizationIn equilibrium denaturation analyses of CA-C using
GdmHCl, dissociation of the dimer into the monomeric intermediate could be
specifically probed by following the intrinsic fluorescence of the only Trp
present (Trp-184). Unfolding of the intermediate did not contribute to the
fluorescence change because Trp-184 is nearly fully exposed to solvent both in
the intermediate and in the denatured protein
(37). The free energy
difference thus determined (
Gud = 12.3
kcal/mol at a 1 M standard state) was much higher than the free
energy of dissociation of the native dimer by simple dilution, as determined
by gel filtration (
Gdis = 6.9 kcal/mol at
1 M standard state). Comparison of the two values allowed
calculation of the relative contributions of quaternary and tertiary structure
changes to the dissociation (or conversely, association) step
(37,
42). For CA-C wild-type, the
value
Gur =
(
Gud
Gdis)/2 was 2.7 kcal/mol and corresponds
to the tertiary reorganization of each CA-C monomeric intermediate molecule
during dissociation of the dimer (or during dimerization)
(37,
42).
We have now likewise determined
Gud for
each interfacial mutant using GdmHCl and fluorescence analysis
(Fig. 5 and
Table I) and compared these
values with the corresponding free energy of dissociation
Gdis =
Ga as determined by gel filtration
(Table I). As expected, the
nine mutants identified as essentially monomeric by gel filtration were found
also monomeric by fluorescence analysis. For these nine mutants, the
fluorescence intensity and maximum emission wavelength at the highest protein
concentrations that could be reasonably used in these experiments (200
µM) corresponded very nearly to those of the monomeric
intermediate found for nonmutated CA-C and did not change substantially at any
GdmHCl concentration (not shown). For each of the 11 alanine mutants that
dimerized, the value
Gur was obtained. The
difference between
Gur for nonmutated CA-C
and each mutant (
Gur) was taken as
a measure of the relative energetic contribution of the truncated side chain
to the tertiary rearrangement of each monomer during dissociation (or
conversely, association) (Fig.
3, gray bars). Only 4 of the 11 interfacial side chains
had no or very little effect on this rearrangement, whereas all others,
irrespective of their role in the establishment of quaternary interactions,
contributed energetically to the rearrangement in a substantial way
(
Gur between 0.8 and 2.3
kcal/mol).

View larger version (19K):
[in this window]
[in a new window]
|
FIG. 5. GdmHCl dissociation of nonmutated CA-C and some mutants at 200
µM followed by intrinsic Trp fluorescence intensity at an
emission wavelength of 350 nm. The curves were fitted to a
dimer-to-monomer two-state transition using Equation 12 in Ref.
37. Circles, parental
CA-C; triangles, mutant K203A; squares, V191A; inverted
triangles, E187A.
|
|
Energetic Contributions of the Interfacial Side Chains to the Folding
Stability of the CA-C MonomerDissociation of the wild-type CA-C
dimer into the folded monomeric intermediate had no effect on the secondary
structure and was invisible by far UV-CD. Thus, the unfolding of the CA-C
monomer could be specifically analyzed in GdmHCl denaturation experiments
followed by far-UV CD (37).
The free energy of unfolding
Gum of the
monomeric form of each CA-C variant mutated at the interface
(Fig. 6 and
Table I) has been now
determined. The difference between
Gum for
nonmutated CA-C and each mutant
(
Gum) was taken as a measure of the
relative energetic contribution of the truncated side chain to the stability
of the isolated monomeric intermediate
(Fig. 3, white bars).
Truncation of eight interfacial residues did not substantially affect monomer
stability. However, 10 other residues destabilized the monomeric intermediate
by 12 kcal/mol each,
2040% of the free energy of folding of
this already weakly stable form. As expected, destabilization of the
intermediate did not correlate with the role of these residues in
dimerization. The nondestabilizing mutations included several polar side
chains (Thr-148, Glu-175, Glu-187, Gln-192, Lys-199, Lys-203). Most of them
were, at least in the dimer, substantially exposed to solvent. Fluorescence
spectroscopy had shown that the side chain of Trp-184 was essentially
solvent-exposed in the monomeric intermediate and its truncation, as expected,
had no effect on its stability. Met-185 did not contribute to stability
either, which was perhaps unexpected if its substantially buried position in
each subunit (as seen in the dimer) is preserved in the monomeric
intermediate. Among the clearly destabilizing truncations were those of the
nonpolar side chains Ile-150, Leu-151, Leu-172, Val-181, or Leu-189, all of
which were also critical for dimerization, and of polar residues Asp-152,
Arg-154, Thr-188, and Asn-193, most of which were not. Interestingly,
truncation of Glu-180 or Ser-178 not only increased dimerization affinity (see
above) but also stabilized the monomeric intermediate by 1.3 or 2.5 kcal/mol,
respectively. Despite the fact that folding and dimerization of CA-C are not
coupled, many interfacial side chains provide a substantial energetic
contribution to both processes.

View larger version (19K):
[in this window]
[in a new window]
|
FIG. 6. GdmHCl denaturation of nonmutated CA-C and some mutants at 200
µM followed by their ellipticity at 222 nm. The curves were
fitted to a unimolecular two-state transition using Equation 13 in Ref.
37. Circles, parental
CA-C; inverted triangles, mutant K203A; triangles, L172A;
squares, S178A.
|
|
 |
DISCUSSION
|
---|
The CA-C dimerization interface is structurally typical of a
protein-protein interface in many respects
(22,
45,
46). It involves as many as 22
residues from each monomer and buries as much as 1846 Å2 of
the solvent-accessible area (if the simplification of a rigid-body interaction
is accepted), two-thirds of which is contributed from nonpolar side chain
atoms that cluster in a large central area of the contact epitope
(Fig. 4A)
(29,
30). In addition, the
energetic dissection of the interface described here has revealed that all of
the hydrophobic side chains buried at the central region of the contact
epitope are critically involved in dimerization, whereas most largely polar
side chains at the interface rim are not.
However, the thermodynamic results have also revealed major differences
with other interfaces in heterocomplexes and homo-dimeric proteins that have
been subjected to complete alanine scanning: (i) As many as nearly half of the
side chains involved in the CA-CA contact epitope are so critically involved
in the energetic epitope that their individual truncation led to essentially
monomeric protein. In sharp contrast, nearly no other contact side chain
showed a significant positive contribution to affinity. (ii) CA-C dimerization
is a very low affinity process when compared with nearly any other
structurally similar interface that has been analyzed by alanine scanning. The
results obtained indicate that such low affinity is partly due to the presence
of several side chains in the contact epitope (Ser-178, Glu-180, Glu-187, and
especially Gln-192) that not only do not favor association but that
individually decrease the association constant up to more than one
order of magnitude. An increase in affinity has been rarely found upon
mutation of residues in other protein-protein interfaces, and when
encountered, this effect was generally very small
(5,
8,
15,
21). The interfacial residues
that negatively affect affinity, and/or the interactions they make, including
possible electrostatic repulsions at opposite sides of the interface rim (see
"Results"), appeared highly conserved in HIV, even more than some
of the critical residues in the energetic epitope. This remarkable
conservation could be explained if those residues were needed for the
conformational stability of the individual partners. However, truncation of
any of the CA-C side chains that impaired association either had no effect or
substantially increased the conformational stability of both the monomeric and
dimeric forms. It is tempting to speculate that interfacial side chains and
interactions that impair CA-CA affinity may have been evolutionarily conserved
because of a selective functional advantage of a low stability and
dimerization affinity of this CA domain for the productive assembly and
maturation of the conformationally flexible HIV capsid
(31,
37). A nonexclusive
explanation is that these residues may be involved in other interactions
needed for completion of the HIV life cycle. Specifically, Ser-178 may be
phosphorylated in HIV. Mutation of this residue affected viral infectivity,
and the evidence suggested that Ser-178 phosphorylation is essential for the
viral uncoating process (47).
Thr is found at the equivalent position in many HIV-1 variants, but this
residue also has the potential to be phosphorylated. Glu-180, Glu-187, and
Gln-192 were all found involved in an alternative intermolecular interface
between the N- and C-terminal domains as observed in crystals of CA of HIV-1
complexed with antibody (31).
The involvement of some residues at the CA-C dimerization interface in the
folding stability of the CA-C monomer, its conformational rearrangement, CA
phosphorylation, and/or possible alternative types of CA-CA interactions
during capsid assembly add strength to the notion that structurally
overlapping functions may be frequent in viral capsids and may impose severe
constraints to virus evolution
(48).
For many protein heterocomplexes or homo-oligomers including CA-C,
protein-protein recognition is not coupled to their folding but involves
association of already folded monomers, in a process that frequently includes
additional conformational rearrangements of the interacting polypeptides. A
molecular interpretation of free energy differences found upon mutation of
residues at protein-protein interfaces thus requires careful consideration of
the specific process that is being observed by the probe used
(49). The combined
thermodynamic analyses carried out for CA-C allowed us to distinguish for each
interfacial side chain its energetic contributions to the folding stability of
the CA-C monomer, to the tertiary conformational rearrangement of the monomers
during dimerization, and to their direct association through the establishment
of quaternary interactions
(42). Many residues in
protein-protein interfaces have their side chains oriented toward the
interacting partner, and it is often assumed that the effects of interfacial
residues on the folding stability of the monomeric subunits are generally
minor or even negligible. In fact, the results with CA-C show that as much as
half of the interfacial side chains can make important contributions to the
stability of the monomeric form. Several of these residues clearly participate
in the hydrophobic core of each subunit as they appear in the dimer structure,
but other residues important for monomer stability have their side chains
oriented toward the interface. Determination of differences in tertiary
structure between the isolated subunit and the oligomer may be needed for a
satisfactory molecular explanation of the thermodynamic results. For CA-C,
this could be achieved by determining the structure of the monomeric W184A
mutant (underway). Because for CA-C the establishment of quaternary
interactions is accompanied by an energetically substantial tertiary
rearrangement of the interacting subunits, it was also important to
differentiate between the contributions of each interfacial side chain to
these processes. Most of the side chains were involved in both, but to widely
diverse extents. For example, Asp-152 and Gln-192 had a positive contribution
to the conformational rearrangement of the monomers during dimerization,
whereas Asp-152 had no significant effect on and Gln-192 actually impaired
this specific process. Even with such combined thermodynamic approach, the
energetic dissection of any protein-protein interface may still be complicated
by nonadditive (cooperative) effects
(49). This is also clearly
seen with CA-C, as individual truncation of each of nearly half of the
interfacial residues led to differences in the free energy of association
large enough to essentially abolish dimerization.
The isolated wild-type CA-C domain has been shown previously to inhibit HIV
assembly (4). Now, alanine
scanning has allowed us to identify the several residues at the CA-C
dimerization interface that do not positively contribute to the stability
and/or association of this small protein domain
(Table I and
Fig. 3). Correctly folded CA-C
variants with substantially increased affinity for wild-type CA could be
obtained by rational or combinatorial mutagenesis of some of these residues,
in particular those that keep dimerization affinity low. CA-C mutant Q192A,
with its 20-fold increased affinity, could serve as a starting point for this
approach to design a high affinity interfacial inhibitor of native CA
dimerization and HIV-1 assembly and infectivity.
 |
FOOTNOTES
|
---|
* This work was supported by grants from Ministerio de Sanidad y Consumo
(Grant FIS 01/0004-01 to M. G. M. and Grant FIS 01/0004-02 to J. L. N), Grants
BIO20000408 from MCyT and 08.2/0008/2000 from Comunidad de Madrid, an
institutional grant from Fundación Ramón Areces (to M. G. M.)
and Grant CTIDIB/2002/6 from Generalitat Valenciana (to J. L. N.). The costs
of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶
To whom correspondence should be addressed. Tel.: 34-91-3978462; Fax:
34-91-3974799; E-mail:
mgarcia{at}cbm.uam.es.
1 The abbreviations used are: HIV, human immunodeficiency virus; SIV, simian
immunodeficiency virus; CA, capsid protein of HIV; CA-C, C-terminal domain of
CA; CD, circular dichroism, GdmHCl, guanidinium hydrochloride. 
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge Prof. C. Alonso for the use of a
spectropolarimeter and M. A. Fuertes for technical help with this
instrument.
 |
REFERENCES
|
---|
- Babé, L. M., Pichuantes, S. & Craik, C. S.
(1991) Biochemistry
30,
106111[Medline]
[Order article via Infotrieve]
- Zhang, Z.-Y., Poorman, R. A., Maggiora, L. L., Heinrikson, R. L.
& Kézdy, F. J. (1991) J. Biol.
Chem. 266,
1559115594[Abstract/Free Full Text]
- Schramm, H. J., Boetzel, J., Buttner, J., Fritsche, E., Gohring,
W., Jaeger, E., Konig, S., Thumfart, O., Wenger, T., Nagel, N. E. &
Schramm, W. (1996) Antiviral Res.
30,
155170[CrossRef][Medline]
[Order article via Infotrieve]
- Lanman, J., Sexton, J., Sakalian, M. & Prevelige, P. E., Jr.
(2002) J. Virol.
76,
69006908[Abstract/Free Full Text]
- Cunningham, B. C. & Wells, J. A. (1989)
Science 244,
10811805[Medline]
[Order article via Infotrieve]
- Lau, F. T. & Fersht, A. R. (1989)
Biochemistry 28,
68416847[Medline]
[Order article via Infotrieve]
- Jin, L., Fendly, B. M. & Wells, J. A. (1992)
J. Mol. Biol. 226,
851865[Medline]
[Order article via Infotrieve]
- Cunningham, B. C. & Wells, J. A. (1993)
J. Mol. Biol. 234,
554563[CrossRef][Medline]
[Order article via Infotrieve]
- Kelley, R. F. & O'Connell, M. P. (1993)
Biochemistry 32,
68286835[Medline]
[Order article via Infotrieve]
- Clackson, T. & Wells, J. A. (1995)
Science 267,
383386[Medline]
[Order article via Infotrieve]
- Schreiber, G. & Fersht, A. R. (1993)
Biochemistry 32,
51455150[Medline]
[Order article via Infotrieve]
- Schreiber, G. & Fersht, A. R. (1995) J.
Mol. Biol. 248,
478486[CrossRef][Medline]
[Order article via Infotrieve]
- Kelley, R. F., Costas, K. E., O'Connell, M. P. & Lazarus, R. A.
(1995) Biochemistry
34,
1038310392[Medline]
[Order article via Infotrieve]
- Castro, M. J. M. & Anderson, S. (1996)
Biochemistry 35,
1143511446[CrossRef][Medline]
[Order article via Infotrieve]
- Dall'Acqua, W., Goldman, E. R., Eisenstein, E. & Mariuzza, R.
A. (1996) Biochemistry
35,
96679676[CrossRef][Medline]
[Order article via Infotrieve]
- Dall'Acqua, W., Goldman, E. R., Lin, W., Teng, C., Tsuchiya, D.,
Li, H., Ysern, X., Braden, B. C., Li, Y., Smith-Gill, S. & Mariuzza, R. A.
(1998) Biochemistry
37,
79817991[CrossRef][Medline]
[Order article via Infotrieve]
- Milla, M. E., Brown, B. M. & Sauer, R. T. (1994)
Nat. Struct. Biol. 1,
518523[Medline]
[Order article via Infotrieve]
- Mateu, M. G. & Fersht, A. R. (1998)
EMBO J. 17,
27482758[Abstract/Free Full Text]
- Dall'Acqua, W., Simon, A. L, Mulkerrin, M. G. & Carter, P.
(1998) Biochemistry
37,
92669273[CrossRef][Medline]
[Order article via Infotrieve]
- Sengchanthalangsy, L. L., Datta, S., Huang, D.-B., Anderson, E.,
Braswell, E. & Ghosh, G. (1999) J. Mol.
Biol. 289,
10291040[CrossRef][Medline]
[Order article via Infotrieve]
- Myers, D. P., Jackson, L., Ipe, V. G., Murphy, G. E. &
Phillips, M. A. (2001) Biochemistry
40,
1323013236[CrossRef][Medline]
[Order article via Infotrieve]
- Bogan, A. A. & Thorn, K. S. (1998) J.
Mol. Biol. 280,
19[CrossRef][Medline]
[Order article via Infotrieve]
- Wang, C. T. & Barklis, E. (1993) J.
Virol. 67,
42644273[Abstract]
- Dorfman, T., Bukovsky, A., Öhagen, A., Hoglund, S. &
Göttlinger H. G. (1994) J. Virol.
68,
81808187[Abstract]
- Borsetti, A., Öhagen, A. & Göttlinger, H. G.
(1998) J. Virol.
72,
93139317[Abstract/Free Full Text]
- Reicin, A. S., Ohagen, A., Yin, L., Hoglund S. & Goff, S. P.
(1996) J. Virol.
70,
86458652[Abstract]
- Momany, C., Kovari, L. C., Prongay, A. J., Keller, W., Gitti, R.
K., Lee, B. M., Gorbalenya, A. E., Tong, L., McClure, J., Ehrlich, L. S.,
Summers, M. F., Carter, C. & Rossmann, M. G. (1996)
Nat. Struct. Biol. 3,
763770[Medline]
[Order article via Infotrieve]
- Gamble, T. R., Vajdos, F. F., Yoo, S., Worthylake, D. K.,
Houseweart, M., Sundquist, W. I. & Hill, C. P. (1996)
Cell 87,
12851294[Medline]
[Order article via Infotrieve]
- Gamble, T. R., Yoo, S., Vajdos, F. F., von Schwedler, U. K.,
Worthylake, D. K., Wang, H., McCutcheon, J. P., Sunquist, W. I. & Hill, C.
P. (1997) Science
278,
849853[Abstract/Free Full Text]
- Worthylake, D. K., Wang, H., Yoo, S., Sunquist, W. I. & Hill,
C. P. (1999) Acta Crystallogr. Sect. D Biol.
Crystallogr. 55,
8592[CrossRef][Medline]
[Order article via Infotrieve]
- Berthet-Colominas, C., Monaco, S., Novelli, A., Sibai, G., Mallet,
F. & Cusack, S. (1999) EMBO J.
18,
11241136[Abstract/Free Full Text]
- Jin, Z., Jin, L., Peterson, D. L. & Lawson, C. L.
(1999) J. Mol. Biol.
286,
8393[CrossRef][Medline]
[Order article via Infotrieve]
- Khorasanizadeh, S., Campos-Olivas, R. & Summers, M. F.
(1999) J. Mol. Biol.
291,
491505[CrossRef][Medline]
[Order article via Infotrieve]
- Kingston, R. L., Fitzon-Ostendorp, T., Eisenmesser, E. Z., Schatz,
G. W., Vogt, V. M., Post, C. B. & Rossmann, M. G. (2000)
Structure 8,
617628[Medline]
[Order article via Infotrieve]
- Li, S., Hill, C. P., Sundquist, W. I. & Finch, J. T.
(2000) Nature
407,
409413[CrossRef][Medline]
[Order article via Infotrieve]
- Lanman, J., Lam, T. T., Barnes, S., Sakalian, M., Emmett, M. R.,
Marshall, A. G. & Prevelige, P. E., Jr. (2003) J.
Mol. Biol. 325,
759772[CrossRef][Medline]
[Order article via Infotrieve]
- Mateu, M. G. (2002) J. Mol.
Biol. 318,
519531[CrossRef][Medline]
[Order article via Infotrieve]
- Valdes, R., Jr. & Ackers, G. K. (1979)
Methods Enzymol. 61,
125142[Medline]
[Order article via Infotrieve]
- Darling, P. J., Holt, J. M. & Ackers, G. K. (2000)
Biochemistry 39,
1150011507[CrossRef][Medline]
[Order article via Infotrieve]
- Sayle, R. A. & Milner-White, E. J. (1995)
Trends Biochem. Sci. 20,
374376[CrossRef][Medline]
[Order article via Infotrieve]
- Vriend, G. (1990) J. Mol.
Graph. 8,
5256[CrossRef][Medline]
[Order article via Infotrieve]
- Neet, K. E. & Timm, D. E. (1994)
Protein Sci. 3,
21672174[Abstract/Free Full Text]
- Janin, J. (1997) Proteins
28,
153161[CrossRef][Medline]
[Order article via Infotrieve]
- Wrobel. J. A., Conrad, M. J., Bloedon, E., Swanstrom, R. &
Hutchison, C. A. (2000) AIDS Res. Hum.
Retroviruses 16,
20492054[CrossRef][Medline]
[Order article via Infotrieve]
- Miller, S. (1989) Protein Eng.
3,
7783[Abstract]
- Jones, S. & Thornton, J. M. (1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1320[Abstract/Free Full Text]
- Cartier, C., Sivard, P., Tranchat, C., Decimo, D. Desgranges, C.
& Boyer, V. (1999) J. Biol. Chem.
274,
1943419440[Abstract/Free Full Text]
- Mateu, M. G. (1995) Virus Res.
38,
124[CrossRef][Medline]
[Order article via Infotrieve]
- Greenspan, N. S. & di Cera, E. (1999)
Nature Biotechnology 17,
936937[CrossRef][Medline]
[Order article via Infotrieve]
- Kraulis, P. J. (1991) J. Appl.
Crystallogr. 24,
946950[CrossRef]