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Previous Article | Next Article 
Journal of Virology, April 2001, p. 3960-3964, Vol. 75, No. 8
Institut für Molekulare Biotechnologie,
D-07745 Jena,1 and Dermatologische Klinik,
Universität Erlangen/Nürnberg, D-91052
Erlangen,2 Germany
Received 9 October 2000/Accepted 17 January 2001
The Nef protein of the simian and human immunodeficiency viruses is
known to directly bind and downregulate the CD4 receptor. Although the
molecular mechanism is well understood, direct binding of Nef and CD4
is difficult to demonstrate and is believed to be of low affinity.
Applying nuclear magnetic resonance and fluorescence spectroscopy, we
biophysically reevaluated the CD4-Nef complex and found the
dissociation constant to be in the submicromolar range. We conclude
that additional, so far disregarded residues in the N terminus of Nef
are important for interaction with CD4.
Human immunodeficiency virus type 1 (HIV-1) Nef is a protein containing roughly 200 amino acid residues. It
is a membrane-associated protein that is produced at the earliest stage
of viral gene expression (13) and is a component of viral
particles (39). Nef has been reported to have diverse
effects on cellular signal transduction pathways. It interacts with
various cellular protein kinases and acts both as a kinase substrate
and as a modulator of kinase activity (7, 19, 22). In
addition, Nef has been demonstrated to downregulate cell surface
receptors CD4 and major histocompatibility complex class I (3, 9,
15, 23, 30, 31, 35). Nef-mediated downmodulation of CD4 is well
understood now and appears to involve a whole set of factors. At least
two distinct motifs in a long loop region of the protein were found to
bind adaptins (AP 1, 2, and 3) (10-12, 18, 28). One of
these motifs was additionally reported to interact with the regulatory
unit of a vacuolar proton pump also involved in CD4 downregulation
(29). The We focused our investigations on direct in vitro binding between
essentially complete binding partners. In particular, we used a
chemically synthesized peptide comprising all 31 C-terminal residues
(403 to 433) of human CD4 with a fluorescein label at its N terminus
and a recombinantly expressed full-length Nef protein from HIV-1 strain
SF2. The CD4 peptide (403 to 433) and other peptides for controls (Fig.
1A) were purchased as
reversed-phase high-performance liquid chromatography-purified products
(Interactiva Biotechnologie, Ulm, Germany, and Jerini Biotools, Berlin,
Germany). Identity was confirmed by matrix assisted laser desorption
ionization time of flight mass spectrometry. Nef was overproduced as a
polyhistidine-tagged fusion protein in Escherichia coli
BL21(DE3) harboring the Nef gene from the HIV-1 isolate SF2 in a pET15b
vector (Novagen) and the plasmid pUBS520 encoding the tRNA
argU (27). Bacteria were grown aerobically at
37°C in Luria broth or M9 medium (38) containing ampicillin (200 µg/ml) and kanamycin (100 µg/ml). Cells were
induced at an optical density at 600 nm of about 0.9 with 1 mM
isopropyl-
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3960-3964.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Direct In Vitro Binding of Full-Length Human
Immunodeficiency Virus Type 1 Nef Protein to CD4 Cytoplasmic
Domain
ABSTRACT
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Abstract
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-subunit of COPI coatomers was shown to bind
Nef subsequently to adaptins and seems to direct CD4 to a degradation
pathway (8, 34). From mutational analysis it is known that
residues 407 to 418 in the cytoplasmic tail of CD4 are necessary and
sufficient for downregulation of CD4 by Nef (1, 4, 14,
37). Three-dimensional structures are known from both the CD4
cytoplasmic domain (40, 41) and Nef proteins with
N-terminal and, in some cases, additional deletions (5, 20,
26). Nuclear magnetic resonance (NMR) investigations into the
interaction between CD4 and Nef using a 13-residue peptide of CD4
(residues 407 to 419) and several Nef mutants (Nef
2-39
and Nef2-39, 159-173) elucidated residues in these
Nef deletion mutants that were affected by binding of CD4(407-419)
(21). The dissociation constant (KD) of this complex, however, was found to be
only in the range of 1 mM. Although N-terminal amino acid sequences
among Nef proteins are not conserved, some residues therein are known
to be essential for downregulation of CD4 expression (2, 24,
25). Moreover, a study employing the yeast two-hybrid system
suggests that residues important for CD4 binding are scattered all over
the Nef sequence (36).
-D-1-thiogalactopyranoside for 2 to 3 h,
harvested by centrifugation (3,000 × g; 4°C) and sonicated in phosphate-buffered saline (PBS) buffer (8.8 mM
Na2HPO4, 0.94 mM
KH2PO4[pH 7.4], 150 mM NaCl). The extract was
clarified by centrifugation for 30 min at 20,000 × g
at 4°C and applied to a TALON metal affinity column (Clontech). After
being washed with 50 mM potassium phosphate buffer (pH 7.5) containing
300 mM NaCl, the protein was eluted with 150 mM imidazol in the same buffer. Fractions containing the fusion protein, as determined by
denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were pooled, desalted, and applied to a butyl Sepharose column (Pharmacia, Uppsala, Sweden) in PBS containing 8.4% (by weight) ammonium sulfate. The bound protein was eluted with a gradient from
8.4% to 0% (by weight) ammonium sulfate in PBS buffer. Fractions containing the fusion protein were pooled, desalted, and concentrated (Macrosep; molecular weight cutoff, 10 kDa; Pall Filtron). The purified
fusion protein was cleaved for 3 to 4 h at room temperature with
0.3 U of biotinylated thrombin (Pharmacia) per mg of protein, yielding
the full-length Nef protein from HIV-1 strain SF2
(MGGKWSKRSMGGWSAIRERMRRAEPRAEPAADGVGAVSRDLEKHGAITSSNTAATNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKAALDISHFLKEKGGLEGLIWSQRRQEILDLWIYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKVEEANEGENNSLLHPMSLHGMEDAEKEVLVWRFDSKLAFHHMARELHPEYYKDC) with an additional eight amino acids at its N terminus
(GSHMLEDP). Thrombin was separated from Nef by
treatment with 15 µl of streptavidin agarose (Novagen) per unit.
After centrifugation, the supernatant was applied to a HiLoad Superdex
75 prep grade column (Pharmacia) and eluted with PBS. All buffers used
in this study were degassed and supplemented with 14 mM
2-mercaptoethanol. Fractions containing Nef protein were pooled and
concentrated. The identity of the protein was confirmed by matrix
assisted laser desorption ionization time of flight mass spectrometry
and N-terminal amino acid sequence analysis.
View larger version (17K):
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FIG. 1.
(A) Overview of N-terminal fluoresceinylated CD4
peptides used for Nef binding studies. Amino acid sequences are given
using the one-letter-code for the CD4 peptides named on the left. In
addition, the residue numbers corresponding to the respective sequence
positions in CD4 are shown in the top line. (B) Fluorescence titrations
of 1.1 µM fluoresceinylated CD4(403-433) ( 
), 1.0 µM
fluoresceinylated CD4(403-419) (
), and as a control, 1.1 µM FITC
isomer I (
) as a function of Nef concentration. Values result from
differences in the fluorescences of CD4 peptides or FITC with PBS
buffer-solved Nef protein and PBS buffer without Nef. Assuming a simple
bimolecular reaction between Nef and the CD4 peptide, analysis by
nonlinear curve fitting yielded KD values of
0.87 and 1.4 µM for CD4(403-433) (solid line) and CD4(403-419)
(dashed line), respectively. (C) Fluorescence titrations of 1.0 µM
fluoresceinylated CD4(407-419) (
) and 1.0 µM fluoresceinylated
CD4(LL-AA) (
) as a function of the Nef concentration. Nonlinear
curve fitting yielded a KD value of 3.3 µM for
CD4(407-419) (solid line). (D) Scatchard plot of the data points shown
in panels B and C for binding of HIV-1 Nef to CD4(403-433) (),
CD4(403-419) (), and CD4(407-419) () peptides. The x
axis (r) corresponds to the ratio of bound Nef to the total CD4 peptide
concentration, and r/cf (y axis) is the ratio of r and
unbound Nef. Linear regression analysis (solid, dashed, and dotted
lines) yielded dissociation constants (
1/KD,
slope) and numbers of Nef binding sites per CD4 molecule (x
axis intercept), the latter being close to 1 in all cases. It can
easily be seen that most data points are very close to the respective
fit curves. Some values for CD4(403-433) are more scattered than data
points derived from other peptides. These experimental difficulties may
be due to the presence of three cysteine residues in CD4(403-433).
We used fluorescence titrations to determine the dissociation constant
(KD) values of full-length Nef and several CD4
peptides (Fig. 1A). Fluorescence measurements were carried out at 298K on an SLM Aminco Fourier Transform Spectrofluorometer 48000 MHF (SLM
Instruments Inc.) and an LS50B (Perkin-Elmer) in standard fluorescence
cells of SUPRASIL quartz glass (10 by 10 mm; Hellma, Müllheim,
Germany). Fluorescence was measured with permanent stirring in PBS
buffer using excitation and emission wavelengths of 495 and 520 nm,
respectively. Appropriate amounts of Nef were added to the fluorescent
component solved in PBS buffer. As a control the same titrations were
performed with buffer devoid of Nef. Figure 1B shows the change of
fluoresceinyl-CD4(403-433) relative fluorescence as a function of the
Nef concentration. Assuming a simple bimolecular reaction between Nef
and CD4, analysis by nonlinear curve fitting (32) yielded
a KD value of 0.87 ± 0.19 µM. As
additional parameters, minimum (0.13 ± 0.26) and maximum
(12.6 ± 0.74) fluorescences were fitted from the experimental data. Both values are in accordance with the experimental data and
indicate the fit to be correct. Fluorescein isothiocyanate (FITC-I)
(Sigma) as a control did not bind to Nef (Fig. 1B). An independent
evaluation employing a Scatchard plot analysis with a linear regression
analysis (Fig. 1D) confirmed the KD value to be
0.84 µM.
The observed KD value for binding of full-length
Nef to CD4(403-433) is about 1,000-fold lower than that observed for
Nef mutants Nef2-39 and
Nef2-39,159-173 and CD4(407-419)
(21). Differences in both studies are in the length of the
CD4 peptide and the completeness of the Nef protein. The possibility
that the C-terminal tail of the CD4 cytoplasmic domain is involved in
Nef binding can be excluded based on all mutation experiments (1,
4, 14, 36, 37). To be sure that our in vitro assay was in
accordance with published data derived from assays in cell cultures, we
determined the dissociation constant of a CD4 peptide C-terminally
truncated at exactly the same position as the peptide used by Grzesiek
and colleagues (21) in their study. The dissociation
constant of 1.4 µM (Fig. 1B and D) obtained for Nef and CD4(403-419)
suggests only a minor role of residues 420 to 433 in CD4 for Nef binding.
In the present study, a CD4 peptide was used that starts at Gln-403,
which builds the N-terminal cap of an 
-helix (40). This
helix cap could not form in the CD4 peptide (residues 407 to 419) used
by Grzesiek and colleagues. Gratton and colleagues (17)
concluded based on their mutational studies that a correlation exists
between the presence of this -helix in CD4 and susceptibility to
downregulation by Nef. All these data suggest that the existence of a
preformed -helix in CD4 supports binding to Nef. To measure the
contribution of the four residues forming the helix cap, we determined
the dissociation constant of CD4 peptide (residues 407 to 419) and
full-length Nef to be 3.3 µM (Fig. 1C and D). Thus, the presence of
residues 403 to 406 forming the helix cap increases the affinity of CD4
to Nef by a factor of roughly 2. The CD4 peptide (407 to 419) yielding
a KD of 3.3 µM for Nef binding has exactly the
same sequence as that used in earlier studies reporting a
KD of 1 mM (21). However, the amino
acid sequence of the Nef protein used in the present study was
complete, in contrast to that used in earlier studies, which lacked 38 N-terminal residues (21). This strongly suggests that an
intact N-terminal region of Nef is important for high-affinity binding
to CD4.
As a final control, we carried out a binding study with Nef and a CD4 peptide (403 to 419) in which leucines 413 and 414 were replaced with alanines (Fig. 1C). This mutation is reported to render CD4 refractory to Nef-induced downregulation (1). No dissociation constant could be determined from the data points measured within the Nef concentration range between 0 and more than 13 µM, suggesting that mutation of leucines 413 and 414 to alanines drastically reduces the affinity of CD4 to Nef. This observation is in perfect accordance with published data (1, 21). It is known from mutational analysis that residues 407 to 418 of CD4 are necessary and sufficient for downregulation of CD4 by Nef (1, 4, 14, 37). Our in vitro studies basically confirm these observations. Residues N- and C-terminal to this part of CD4, however, seem to have a minor but significant contribution to Nef binding.
To ultimately elucidate details of binding of Nef to CD4, knowledge of
the three-dimensional complex structure is desired. To pursue this
long-term goal and to confirm high-affinity binding of Nef and CD4 by
an additional method, we employed NMR spectroscopy. Observation of
chemical shift changes in a protein upon ligand binding is a sensitive
method for measuring the strength of an interaction and for defining
the protein's interaction surface (16, 33). Especially
useful and sensitive are, for example, heteronuclear single quantum
coherence (HSQC) spectra. Thus, 1H, 15N HSQC
spectra of 15N-labeled full-length Nef protein with
increasing amounts of CD4(403-433) peptide were recorded. Titrations
were performed in quarter steps in respect to the molar ratio of the
Nef protein and the CD4 peptide. From a KD of
about 1 µM or even below, dissociation rates of less than 100 Hz can
be expected even in the case of diffusion-controlled association. Thus,
exchange between free and CD4-bound Nef should be slow on the NMR
chemical shift time scale at least for some of the 1H,
15N amide cross resonances. Indeed, intensities of some of
the amide resonances in the 1H, 15N HSQC
spectra decreased without shifting while new resonances appeared with
increasing intensities during ongoing titration with CD4 peptide (Fig.
2). Assuming that the
resonance pairs shown in Fig. 2B and C belong to the same amide
cross-resonances of Nef in the presence and absence of the CD4 peptide,
their proton chemical shift distances of 510 and 330 Hz, respectively,
indicate that exchange between bound and unbound Nef is significantly
slower than 300 Hz. A number of other resonances (one can be seen in Fig. 2C) shifted during titration with the CD4 peptide up to 30 Hz,
suggesting the dissociation of the complex to be fast compared to this
time scale. Both observations confirm that the dissociation rate of the
complex is about 100 Hz and, given that the association rate is
diffusion controlled (<108 Hz M
1), the
resulting dissociation constant is 1 µM or less, which is in perfect
agreement with the results from the fluorescence titration. Most of the
amide resonances in the HSQC spectra did not show significant changes
(Fig. 2A), indicating that the overall three-dimensional structure of
Nef does not change dramatically upon binding of CD4. Because
assignment of resonances of the Nef variant (SF2) used in this study
was not sequence specific, it is not possible at the present stage to
directly identify Nef residues involved in CD4 binding.
|
It is worth mentioning that our study was carried out with the CD4 peptide and Nef protein not anchored on the same side of a membrane as would be the case in vivo. Thus, in a living cell, binding affinity between Nef and CD4 can be expected to be even greater than observed in our in vitro system due to a much more favorable entropic term of the binding energy.
Employing a synthetic CD4(403-433) peptide and the recombinant full-length Nef protein purified by a novel highly native procedure under reducing and oxygen-excluding conditions, we were able to show high-affinity binding between HIV Nef and CD4. The resulting complex may be suitable for high-resolution structure determination by NMR spectroscopy or X-ray crystallography. Although this will take significant additional efforts, it will yield better insight into interference with host signal transduction proteins by viral proteins. Exploring new target proteins in HIV infection, aside from reverse transcriptase and protease, is becoming increasingly important. The fluorescence assay for measurement of CD4-Nef binding described in our study is possibly suitable to be employed directly or in a modified form for high-throughput screening assays to find substances that interfere with CD4-Nef interaction.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to D.W. and A.S.B. (SFB 466, A4, and B1).
We are grateful to R. Mattes (Institut für Industrielle Genetik, Universität Stuttgart) for providing the plasmid pUBS520. We thank K.-H. Gührs and B. Schlott (Institut für Molekulare Biotechnologie, Jena, Germany) for carrying out mass spectroscopy and N-terminal amino acid sequencing.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Molekulare Biotechnologie, Beutenbergstr. 11, 07745 Jena, Germany. Phone: 49 3641 656440. Fax: 49 3641 656444. E-mail: willbold{at}imb-jena.de.
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