Fischer W.B. Viral Membrane Proteins: Structure, Function, and Drug Design
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2.9. Summary of the CD4–Nef Interaction
Residues 407–419 of CD4 were shown to be important for Nef binding by mutation
experiments. This was confirmed by direct in vitro binding experiments. Furthermore, the
presence of helical secondary structure in the amino-terminal part of CD4(407–419) seems to
increase affinity to Nef. On the other side of the complex, residues W57, L58, E59, G95, G96,
L97, R106, and L110 of Nef and some not yet identified parts of the amino-terminal part of
Nef are important for binding to CD4.
It is important (especially for the scope of this book) to mention that, so far, all studies
were carried out with CD4 peptide and Nef protein not anchored on the same side of a mem-
brane as it would be the case in vivo. Thus, in a living cell, binding affinity between Nef and
CD4 can be expected to be even higher than observed in the above described in vitro studies
due to a much more favorable entropic term of the binding energy.
3. Interaction of Nef with Human Lck
Nef has been reported to have diverse effects on cellular signal transduction pathways
(Wolf et al., 2001). It interacts with various cellular protein kinases and acts both as a kinase
substrate and as a modulator of kinase activity (Greenway et al., 1996; Baur et al., 1997;
Harris, 1999). A highly conserved proline repeat motif PxxP was found in all Nef variants
(Saksela, 1997), which is known as the minimal consensus sequence defining the ligands of
SH3 domains (Cicchetti et al., 1992; Ren et al., 1993). Binding of Nef to various members of
the Src family of protein kinases, namely Hck and Lyn (Lee et al., 1995; Saksela et al., 1995),
Lck (Collette et al., 1996; Greenway et al., 1996), Fyn (Greenway et al., 1996), and Src
(Arold et al., 1998) has been demonstrated.
3.1. Lymphocyte Specific Kinase Lck
Protein tyrosine kinases are involved in signal transduction pathways that regulate cell
growth, differentiation, activation, and transformation. Human lymphocyte specific kinase
(Lck) is a 56-kDa protein involved in T cell- and IL2-receptor signaling. Lck is a typical
member of the Src-type tyrosine kinase family and consists of four functional domains,
namely unique, SH3, SH2, and kinase. Whereas amino acid sequences of the other domains
are highly conserved among different kinases, those of the unique domains are not. Lck
unique domain is thought to serve as a membrane anchor, but also plays a role in function and
specificity of the other domains, for example, SH2 and SH3 (Carrera et al., 1995). Because
of its key role in T cell signaling and activation (Isakov and Biesinger, 2000), it is not
surprising that pathogenic factors like HIV and Herpesvirus saimiri have evolved effector
molecules that target Lck to ensure their own replication and persistence. In particular,
HIV-1 Nef and H. saimiri Tip proteins directly bind to Lck SH3 domain. Tip binding to Lck
SH3 domain was shown to be based on additional contacts to those typically observed for
poly-proline peptides bound to SH3 domains (Schweimer et al., 2002).
While Nef binding to most other SH3 domains assayed so far was shown to be based
on K
D
in the micromolar range (Arold et al., 1998), Nef affinity to Hck SH3 was remarkably
higher (Lee et al., 1995; Arold et al., 1998).
278 Dieter Willbold
3.2. X-ray Structures of Nef–SH3 Complexes
Several X-ray crystallographic studies have revealed the complex structures of
so-called core-Nef (Nef
2–39,159–173
) with Fyn and Hck SH3 domains (Lee et al., 1995; Arold
et al., 1997, 1998). Residues 71–77 (the poly-proline helix), Lys82, Ala83, Asp86, Leu87,
Phe90, Trp113, Thr117, Qln118, and Tyr120 of Nef core domain were identified to be impor-
tant for binding to Fyn SH3 domain. Because at least for HIV replication in T helper cells,
Nef interaction with Lck certainly is more relevant than with Hck, and because Lck is
associated with CD4, which in turn binds Nef, too, investigations to map the Lck–Nef-
interaction site were of particular interest.
3.3. NMR Spectroscopy is a Suitable Tool to Map
Nef–Lck Interaction Sites
A study to map the full-length Nef interaction site on Lck was recently carried out by
NMR spectroscopy (Briese et al., 2004). Because significant differences between core-Nef
and full-length Nef proteins were already demonstrated for binding to CD4 cytoplasmic
domain (Preusser et al., 2001), full-length Nef was used for this study, too. Further, to inves-
tigate any contribution of the Lck unique domain to Lck–Nef interaction, a recombinant
protein consisting of the 120 amino-terminal residues of Lck comprising the unique and SH3
domains of Lck (LckU3) was used. Although the unique part of LckU3 was shown to be
absent of any stable structural elements (Briese and Willbold, 2003), it seemed possible to be
involved in Nef binding, because Lck uniquely is responsible for Lck binding to human CD4
receptor cytoplasmic domain (Veillette et al., 1988; Shaw et al., 1990; Turner et al., 1990;
Huse et al., 1998; Lin et al., 1998), which in turn binds directly to Nef (Grzesiek et al., 1996;
Preusser et al., 2001, 2002).
To elucidate the details of Nef binding to Lck(1–120), chemical shift perturbation map-
ping by NMR spectroscopy was employed, as already described in the section “High affinity
between CD4(403–433) and full-length Nef can be confirmed by NMR spectroscopy.” Thus,
1
H-
15
N-HSQC spectra of
15
N-labeled Lck(1–120) with increasing amounts of full-length Nef
protein were recorded (Figure 18.6). In contrast to the above-described NMR titration study
with Nef and CD4 cytoplasmic domain, all resonances of LckU3 are known (Briese et al.,
2001). Therefore, it was possible to identify those LckU3 residues involved in Nef binding
(Briese et al., 2004).
From a reported dissociation constant, K
D
, of about 10 M (Arold et al., 1998), disso-
ciation rates of several hundred up to 1,000 Hz can be expected in the case of a diffusion
controlled association rate. Thus, exchange between free and Nef-bound Lck were expected
to be fast or intermediate on the NMR chemical shift time scale for
1
H-
15
N amide cross-
resonances of the affected residues. Indeed, a small number of resonances shifted during titra-
tion with Nef while some other resonances disappeared with increasing Nef concentration.
Both observations confirm that the dissociation rate of the complex is within the expected
region of several hundred hertz and, given the association rate to be diffusion controlled
(10
8
HzM
1
), the resulting dissociation constant is 10 M or less, which is in perfect
agreement with previously reported results (Arold et al., 1998).
Interaction of HIV-1 Nef with Human CD4 and Lck 279
3.4. The Unique Domain of Lck is Not Involved in Nef Binding
Most of the amide resonances in the HSQC spectra did not show significant changes
indicating that the overall three-dimensional structure of Lck(1–120) does not dramatically
change upon Nef binding. More importantly, no residue of the unique domain is affected by
280 Dieter Willbold
Figure 18.6. Mapping of LckU3 residues involved in HIV-1 Nef binding. Selected region (A) of superimposed
1
H-
15
N-HSQC spectra of 15N-isotope labeled LckU3 in absence (gray contour lines) and presence (black contour
lines) of full-length HIV-1 Nef protein (Briese et al., 2003). During titration, some peaks did neither shift nor
disappear (e.g., side-chain ε-imino groups of Trp13 and Trp98, backbone amide groups of Ala68, Ile90, and Leu103),
some peaks disappeared completely or to a large extent (e.g. side chain ε-imino group of Trp97, backbone amide
groups of Leu69, Asp77, Leu80, Leu91, Trp98, Lys99, and Ala100), and some peaks shifted (e.g., backbone amide
groups of Leu69). NMR-samples contained 192 M uniformly
15
N-labeled LckU3 in PBS buffer with 10% D
2
O. At
the titration end point, the samples contained a 1.5-fold molar excess of Nef protein. All NMR spectra were recorded
at 298 K on a Varian Unity INOVA spectrometer working at 750 MHz proton frequency. Amino acid sequence and
domain representation of LckU3 (B) with the unique and SH3 domains indicated as horizontal bars. Sequence
position is given by numbers at the bottom. Locations of -sheet secondary structure elements are indicated by
horizontal arrows. Residues of LckU3 that disappeared or shifted upon addition of full-length Nef proteins are
indicated with vertical bars at the appropriate sequence position and the respective letters in the amino acid sequence
are highlighted in bold italic letters.
Interaction of HIV-1 Nef with Human CD4 and Lck 281
Nef binding (Briese et al., 2003). This is interesting, because the unique domain is essential
for Lck–CD4 interaction, and the question whether dissociation of Lck and CD4 is induced
upon Nef binding is controversially discussed (Salghetti et al., 1995; Gratton et al., 1996).
The NMR shift perturbation study clearly shows that residues of the unique domain are not
required for or involved in Nef binding. Therefore, disregarding potential downstream events,
binding of Lck to CD4 and Nef does not seem to be competitive. Previously, based on muta-
tional analysis, non-overlapping binding sites were identified for Lck and Nef on CD4
(Gratton et al., 1996).
3.5. Mapping of the Nef Interaction Site on Lck SH3
LckU3 residues that are affected upon Nef binding (Figure 18.7) are essentially those
reported in similar investigations of SH3 domain interactions with poly-proline peptides
(Larson and Davidson, 2000), and are in general accordance (Tyr72, Ser75 to Asp79, Glu96,
Trp97, Asn114, and Phe115) with the analysis of the crystallographic complex structure of
Fyn SH3 domain with Nef deletion variant Nef
2–39,159–173
(Arold et al., 1997, 1998). Fyn
and Lck SH3 domains show a sequence identity of 50% (Arold et al., 1997). Differences
between Fyn-SH3–Nef
2–39,159–173
(Arold et al., 1997) and Lck-SH3–Nef (Briese et al.,
2003) are observed in that residues Ile67, Leu69, Glu73, Leu91, Gln93 to Gly95, Trp98 to
Ala100, Phe110, Ile111, Phe113, and Ala117 seem to be affected upon Lck SH3 domain
complex formation with full-length Nef but the respective homolog residues in Fyn SH3 were
not identified to be important for interaction with Nef
2–39,159–173
. Only His70 (homolog to
Tyr91 of Fyn SH3), which was identified to play a role for Fyn-SH3–Nef
2–39,159–173
inter-
action, was not found to be involved in binding of full-length Nef to Lck-SH3 in the present
study. Pro112 (homolog to Pro134 of Fyn SH3), which also was described to be involved in
Fyn-SH3–Nef
2–39,159–173
interaction, was not detectable in the HSQC titration experiment
we used to map the interaction surface, due to the lack of an observable amide proton in
proline residues. These differences between both studies may be due to the use of full-length
Nef in the present study compared to a Nef deletion variant (Nef
2–39,159–173
) used in the
X-ray analysis (Arold et al., 1997). It may also reflect a higher sensitivity of the NMR
Figure 18.7. (A) Visualization of the Nef binding site on Lck SH3 domain as ribbon diagram and (B) surface view.
Dark regions represent those residues that were affected by binding of full-length Nef to LckU3 (Figure 18.6B).
The figures were prepared using MOLMOL (Koradi et al., 1996).
titration approach because it also detects indirectly induced changes in Lck SH3 domain
conformation upon Nef binding. In any case, however, both studies agree that the inter-
action between Nef and Src-type tyrosine kinase SH3 domains is basically of a canonical
SH3–poly-proline type.
3.6. Summary of the Lck–Nef Interaction
Residues 71–77 (the poly-proline helix), Lys82, Ala83, Asp86, Leu87, Phe90, Trp113,
Thr117, Qln118, and Tyr120 of Nef core domain were identified to be important for binding
to Fyn SH3 domain (Arold et al., 1997). Lck residues that are involved in Nef binding could
be mapped to those typically found in poly-proline peptide binding. The unique domain of
Lck was not affected upon Nef binding (Briese et al., 2004). Whether this is different for
myristylated Nef and myristylated and palmitylated Lck proteins anchored on the same side
of a membrane, remains to be investigated. Thus, in a living cell binding affinity between Nef
and Lck can be expected to be even larger than observed in our in vitro system due to a much
more favorable entropic term of the binding energy. Also, Lck may exert a different binding
behavior to Nef when CD4 is attached to its unique domain via a zinc ion.
Additional efforts will certainly yield better insight into interference of host signal
transduction proteins by viral proteins. Exploring new drug target proteins in HIV infection
aside from reverse transcriptase and protease is becoming increasingly important. Nef could
be a very interesting target.
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286 Dieter Willbold
␣-sarcin 80, 81, 234, 240
accessory gene product 148
acetylcholinesterase 124
activation 102, 105
3A, picornavirus 83
A38L glycoprotein 86
Alphavirus 207, 209, 216, 217, 233, 238,
241, 242
6K 83, 233
lytic cycle 238, 239
amantadine 26, 86, 87, 102–105, 108, 109, 114,
120, 132, 199, 207, 226, 227, 249
mode of action 119
amiloride 86, 190, 192, 214, 215
cyclohexamethylene 190, 192, 197
derivatives 86
dimethyl 190
aminopeptidase N receptors 51
amphiphilic helix 123
anesthetics, local 191
anion channel 118
antiviral 102, 207
activity 87
agents 87
aphids 10, 12
aromatic residues 234, 240
artificial lipid bilayers 207–209, 211–215, 219, 220,
223–225, 228
atebrin 227
AUTODOCK 190, 191, 196
avian influenza 248, 257
2B, picornavirus 83
Baculovirus 121
Barmah forest virus (BFV) 217
benzodiazepine receptor, peripheral mitochondrial
120, 123
beta transducin-repeat containing protein 156
bilayer 178, 182
biological half-life 197
blocker 190
BM2 118, 124
Brownian dynamics 198
budding pore 125
Burnet, MacFarlane 247
C12E8 (detergent) 125
caveolin 8
CCCP (carbonyl cyanide m-chlorophenyl-
hydrazone) 227
CD4 35, 87, 150, 189, 200, 210, 211
degradation 154
gp160 156
CEACAM receptors 52
cell-to-cell movement 3, 4, 5, 7, 14
cellulose acetate electrophoresis 251
channel 177, 178, 181
channel blocker 26
channel-blocking drugs 214, 216
CHAPS 121, 211
chemiosmotic gradient 84
chemokine receptor 38
chlorella 21
Chlorovirus 21
cholesterol 113, 117, 119ff.
cholesterol-binding motif 113
cis 208, 209, 212–214, 219, 220, 224–227
CM2 98
co-entry 79
coiled coil 36
Colman, Peter 252, 254, 257, 259
conductance 207, 209, 212, 213, 215, 225, 228
single-channel 118
coreceptor 35
Coronaviruses
antigenic groups 51, 52
assembly 56
E83
entry inhibitors 59, 60
genomes 55
membrane fusion 54–56
structure 49–51
receptors 51, 52
reverse genetics 57, 58
COS cells 213
covariance matrix 193
CRAC motif 120, 123, 124
Cs
26
C terminus 22
CXCR4 and CXCR5 35, 87, 88
cytoskeleton 5, 8, 122
Index
287