Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange

ArticleinJournal of Virology 84(19):10311-21 · October 2010with22 Reads
Impact Factor: 4.44 · DOI: 10.1128/JVI.00688-10 · Source: PubMed

The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.


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JOURNAL OF VIROLOGY, Oct. 2010, p. 10311–10321 Vol. 84, No. 19
0022-538X/10/$12.00 doi:10.1128/JVI.00688-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Local Conformational Stability of HIV-1 gp120 in Unliganded
and CD4-Bound States as Defined by Amide
Hydrogen/Deuterium Exchange
Leopold Kong,
Chih-chin Huang,
Stephen J. Coales,
Kathleen S. Molnar,
Jeff Skinner,
Yoshitomo Hamuro,
and Peter D. Kwong
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 20892
; Sir William Dunn School of Pathology, Oxford University, Oxford OX1 3RE, United Kingdom
ExSAR Corporation, 11 Deer Park Dr., Suite 103, Monmouth Junction, New Jersey 08852
; and
Bioinformatics and Computational Biosciences Branch, Office of Cyber Infrastructure and
Computational Biology, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892
Received 30 March 2010/Accepted 15 July 2010
The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional
changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states,
meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer
domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints
as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrom-
etry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound
states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydro-
gens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate
of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange
correlated well with alterations in binding surface (P value 0.0004). For the structurally variable gp120,
however, reductions in flexibility extended outside the binding surface, and regions of expected high structural
diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state
than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither
unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse confor-
mations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for
understanding how local conformational stability influences entropic change, conformational diversity, and
structural rearrangements in the gp120-CD4 binding reaction.
Entry by HIV type 1 (HIV-1) into host cells is mediated by
the HIV-1 trimeric viral spike, which is composed of three
gp120 exterior envelope glycoproteins attached noncovalently
to three gp41 transmembrane moieties (reviewed in reference
44). The mechanism of entry is in many ways typical of type 1
viral fusion machinery, with gp120 binding to host receptors on
the cell surface and gp41 rearranging into a trimeric coiled-
coil, thereby promoting a fusion of viral and host cell mem-
branes (reviewed in reference 8). The HIV-1 envelope glyco-
proteins, however, utilize a distinct sequence of receptor
binding and conformational change involving CD4, substantial
CD4-induced structural rearrangements, and coreceptor
CCR5 or CXCR4 (9, 15, 43).
The crystal structures of an unliganded gp120 core from the
closely related simian immunodeficiency virus (SIV), of vari-
ous HIV-1 gp120-CD4 complexes, of various HIV-1 gp120-
antibody complexes, and of the final postfusion HIV-1 gp41
coiled-coil have been determined (5–7, 23, 29, 30, 38, 42, 49).
Structural characterization of the HIV-1 viral spike, mean-
while, is currently available only by modeling or with cryoelec-
tron tomograms (31, 35), the latter for both unliganded, CD4-
bound, and antibody b12-bound states of HIV-1. These studies
reveal that gp120 is composed of two domains (inner and
outer), as well as a bridging sheet minidomain, which forms in
the presence of CD4. The gp120 outer domain appears to be
structurally constant, whereas the gp120 inner domain (and
bridging sheet) displays extensive structural diversity (see Fig.
S1 in the supplemental material).
The conformational stability of the HIV-1 gp120 envelope
glycoprotein, or its flip side, flexibility, has been the subject of
considerable interest and debate. Interest relates to the central
role that structural rearrangements and conformational diver-
sity of gp120 play in HIV-1 entry and immune evasion. Debate,
meanwhile, has centered on the nature of the unliganded state.
Atomic-mobility values (B values) from crystallographic anal-
ysis provide hints, as do thermodynamic measurements of
CD4- and antibody-induced transitions and glutaraldehyde
cross-linking coupled to antigenic analysis (6, 27, 37, 46). Ul-
timately, however, a spatially resolved description of gp120
conformational stability has been lacking.
To define the local conformational stability of HIV-1 gp120,
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID/NIH, 40 Convent Drive, Building 40, Room 4508, Bethesda,
MD 20892-3027. Phone: (301) 594-8439. Fax: (301) 480-0274. E-mail:
† Supplemental material for this article may be found at http://jvi
Published ahead of print on 21 July 2010.
Page 1
we used amide hydrogen/deuterium exchange (HDX) coupled
to mass spectrometric analysis. Digestion by pepsin allowed us
to measure the exchange frequencies for gp120 and CD4 at an
average resolution of 16 residues and covering more than 95%
of the sequences of core gp120 and two-domain CD4. Frag-
ment-specific exchange rates were processed into free energies
of conformational stability and mapped onto published crystal
structures. The results provide a spatially resolved view of local
conformational stability for HIV-1 gp120 and CD4 in unligan-
ded and complexed states.
Protein purification. The HIV-1 gp120 YU2 core construct was prepared as
previously described (43). Constructs were expressed in Drosophila Schneider 2
cells under an inducible metallothionein promoter. The two-domain CD4 was
produced in Chinese hamster ovary cells (39). Preparations of unliganded gp120,
free CD4, and complexed gp120-CD4 were obtained by following procedures
previously described (28). Briefly, the supernatant was collected 2 days after
transfection and passed through an F105 affinity column. Glycans were removed
by digestion with endoglycosidases H and D (Endo H and Endo D) to leave only
the protein-proximal N-acetylglucosamine and 1,4-fucose residues. The two-
domain CD4 was added to create the complex. Deglycosylated gp120 and the
gp120-CD4 complex were passed through a concanavalin A column to remove
gp120 with uncleaved glycans. All samples were further purified by gel filtration
(Hiload 26/60 Superdex S200 prep grade; Amersham) and concentrated to 1
mg/ml in 30 mM phosphate (pH 7.4)–0.35 M NaCl.
Surface plasmon resonance. The kinetic constants of gp120 binding to CD4
were measured by a Biacore 3000 surface plasmon resonance spectrometer as
described previously (49). Briefly, we immobilized four-domain CD4 onto a CM5
sensor chip to a density of 500 response units with amine coupling. We passed
serial dilutions of deglycosylated or glycosylated gp120 from 400 to 3.125 nM
over the chip at 30 l/min for 5 min; this was followed by a 5-min dissociation
phase. The buffer used in the experiments consisted of 10 mM HEPES (pH 7.4),
150 mM NaCl, 3 mM EDTA, and 0.01% surfactant P-20. Sensorgrams were fit
globally with BiaEvaluation 4.1 using a 1:1 binding with drifting baseline model so
that the
values of the fits were all less than 1% of the maximum number of
response units.
Amide hydrogen/deuterium exchange. The amide hydrogen/deuterium ex-
change reaction was initiated by mixing 10 l of gp120 YU2 core, two-domain
CD4, or complex solution with 10 l of deuterated water at 4°C. At five time
points between 15 and 1,500 s of exchange, 30 l of 8 M urea–1 M Tris(2-
carboxyethyl) phosphine hydrochloride (TCEP-HCl) was added to quench the
exchange reaction and reduce the disulfide bonds in the proteins for 1 min at 1°C.
Then, 45 l of the mixture was injected into the ExSAR system (19), which is
kept at 1°C. In the ExSAR system, the sample mixtures were passed through a
pepsin column (bed volume, 104 l; flow rate over the column, 200 l/min) and
the peptic fragments were separated by high-performance liquid chromatogra-
phy (HPLC; C
, a linear gradient of 12% to 33% acetonitrile at a flow rate of
10 l/min in 23 min). The eluted peptic fragments were analyzed by a LCQ mass
spectrometer (Thermo Fisher).
The deuterium incorporation, D(%), into each peptic fragment for each time
point was calculated to take into account the possibility of hydrogen back-
exchange during the passage through the ExSAR system (48) by the formula
D(%) (m m0%)/(m100% m0%), where m is the centroid mass of a
fragment after amide hydrogen/deuterium exchange, m0% is the centroid mass
of the fragment from nondeuterated sample, and m100% is the centroid mass of
the fragment from the fully deuterated sample. Fully deuterated sample was
prepared by mixing 30 l of protein solution with 30 l of 100 mM TCEP-HCl
in deuterated water, followed by overnight incubation at 60°C. Nondeuterated
samples were prepared by mixing 10 l of protein solution with 10 l of water.
Digestion/separation optimization and identification of peptic fragments.
Prior to the submission of unliganded gp120, free CD4, and complexed gp120-
CD4 to HDX analysis, the digestion conditions of the proteins and the separa-
tion conditions for the peptic fragments were optimized using nondeuterated
protein samples (19). In that process, several parameters, including type of
protease, denaturation/reduction conditions, and digestion time, were varied to
generate ideal sequence coverage and special resolution of the proteins. The
peptic fragments were identified by tandem mass spectrometry (MS/MS). Briefly,
nondeuterated protein samples were digested and analyzed by MS in the data-
dependent MS/MS mode. Nonglycosylated fragments were identified by the
SEQUEST search algorithm (45) (Thermo Fisher) using the gp120 YU2 core
and two-domain CD4 sequences. Glycosylated peptides were identified manually
using the MS/MS fragmentation patterns and cleavage site information. All the
results were pooled, and the quality of the peaks was assessed in HDExpress (18).
Data analysis. The frequency of deuterium incorporation for each peptic
fragment of gp120 and CD4 at each time point was measured twice to ensure
reproducibility. The averaged frequency of peptic fragments was mapped onto
the protein sequence for analysis. Distributions of exchange rates were obtained
from these averages using a maximum entropy method (47). The weighted
average exchange rate for each peptide was calculated over the distribution of
exchange rates (34). Each average rate was used to normalize the average
theoretical exchange rate of the same peptide in a completely disordered state.
The theoretical exchange rates were determined by calculations that take into
account the primary sequence of the peptide fragment (3). This normalized
value, or protection factor, is the equilibrium constant for the transition of an
ensemble of amide hydrogen-deuterium exchangeable conformations to an en-
semble of totally exchange-protected conformations (14, 26, 40). The free energy
of conformational stability for each fragment was calculated by substituting
protection factor for the equilibrium constant (K) in the equation G
RTln(K), where G is the free energy of conformational stability, R is the gas
constant, and T is temperature.
The free energies of conformational stability for gp120 and CD4 were mapped
onto the crystal structures of free CD4, unliganded SIV gp120, and complexed
CD4-gp120. Since there were no crystal structures of unliganded HIV-1 gp120,
we threaded the sequence of HIV-1 YU2 core into the structure of unliganded
SIV gp120 using DeepView/SWISS-MODEL and Swiss-PdbViewer (16).
The structure of CD4-bound gp120 was used to fill the disordered residues in the
unliganded structure (residues 220 to 228 using SIV gp120 numbering) and the
V4 loop. Residues characterized in the SIV gp120 crystal structure but not in
HIV-1 gp120 were omitted in the final model. While the unliganded HIV-1
gp120 structure might be better represented by an ensemble of conformations,
we used the core SIV structure for clarity and also because it represents at least
one crystallographically defined conformation within the ensemble. Also, com-
pared to other gp120 structures, the SIV structure better illustrates the potential
for large conformational changes (see Fig. S1 in the supplemental material).
The free energies of conformational stability were compared to the accessible
surface area, root mean square deviation, average temperature factors, fre-
quency of residues within secondary structures, and frequency of potential hy-
drogen bonds to backbone amides for each peptic fragment. These structural
statistics were obtained from crystal structures of gp120 and CD4 using the CNS
(4), the CCP4 (1), and the HBPLUS Hydrogen Bond Calculator (36) software
packages. All comparisons were evaluated through linear regression analysis in
GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA)
and Microsoft Excel.
Explicit incorporation of complex dissociation into HDX analysis. With a
dissociation constant (K
)of200 nM for core gp120 and CD4, at equilibrium,
approximately 10% of a 1-mg/ml equimolar mixture of gp120 and CD4 separates
into uncomplexed, free components. In addition, HDX procedures entail a 2-fold
dilution into deuterated solvent, resulting in additional dissociation. A precise
HDX analysis of the gp120-CD4 complex therefore requires explicit consider-
ation of complex dissociation.
Prior to dilution, the equilibrium concentration of the gp120-CD4 complex can
be calculated from the K
. After dilution, protein fractions approach equilibrium
according to the rate law:
d/dtcomplex k
complex k
gp120兴关CD4 (1)
Substituting [gp120] for [CD4],
d/dtcomplex k
complex k
For initial concentrations, [complex]
and [gp120]
, and conservation of mass,
complex gp120 (3)
Rearranging equation 3, we obtain for [gp120]
gp120 complex
complex (4)
Substituting equation 4 into equation 2 results in
d/dtcomplex k
complex k
This can be rearranged for ease of integration:
Page 2
complex y1
complex y2
y1 y2k
The solution to this integral is:
y2y1 complex
y1 y2t
y1y2 complex
y1 complex
y1 y2t
y2 complex
Thus, by defining k
and k
as well as the concentrations of gp120 and the
complex, changes in the fractional concentrations of the complex during ex-
change reactions can be explicitly determined—as we have done for the complex
data for gp120 and CD4.
Statistically valid methods of HDX error determination: use of duplicate
measurements and overlapping peptides. To define the statistically significant
differences in local conformational stability, appropriately defined measures of
experimental uncertainty were developed. Maximum entropy methods intro-
duced non-Gaussian distributions of rates, which resulted in average extracted
rates that did not have meaningful standard deviations. We therefore attempted
to develop a scheme that retained measures of uncertainty through a maximum
entropy method.
One potential scheme (scheme 1) takes advantage of overlapping fragments in
the HDX analysis (see Fig. S2A in the supplemental material). HDX data from
all 38 peptic fragments of gp120 were processed into free energies of conforma-
tional stability, which were then grouped into the 16 peptic-fragment regions
covering gp120, and averages and standard errors of the mean were calculated
(see Table S1 in the supplemental material). While this scheme produces statis-
tically valid measurements of uncertainty, only 13 of the 16 peptic-fragment
regions contained overlapping fragments. In addition, maximum entropy analysis
failed to generate rate distributions with five of the fragments.
A second scheme (scheme 2) relying strictly on the standard deviation gener-
ated by the repeat experiment was utilized (see Fig. S2B in the supplemental
material). Maximum entropy methods using a common or shared standard de-
viation for each fragment calculated from averaged data were used to extract rate
distributions from nonaveraged exchange curves for 16 peptic fragments that
spanned all 16 regions. This resulted in two exchange rates for each peptic
fragment. The two rates were then processed into free energies of conforma-
tional stability and combined as averages with standard errors of the mean (Table
1). This scheme produced measures of uncertainty for all 16 peptic regions.
However, uncertain measurements were generated from a sample size of two.
Also, because maximum entropy calculations for both replicates were carried out
with the same standard deviation, they were not strictly independent. Compar-
isons, however, between rates (and errors) calculated by the two error analysis
schemes had an R
of 0.99, indicating that the results from the second scheme
could be used with confidence.
NAME. We sought to process the HDX data for gp120 and CD4 into absolute
measures through processes which involved global consideration of experimental
time points and the derivation of statistically valid measures of uncertainty. To
this end, we derived and utilized a method of normalized assessment of maxi-
mum entropy (NAME). NAME entails calculation of free energies of confor-
mational stability from protection factors, which are the exchange rates of an
unstructured peptide normalized by the observed exchange rates of the same
peptide in the protein structure. The observed rates for peptic fragments are the
averages of exchange rate distributions generated by the maximum entropy
method from the HDX data (48). The theoretical rates are generated from
software for each backbone amide site.
Methodological alterations and overall scheme. Standard
procedures for HDX analysis (19, 22) were followed, although
extensive gp120 glycosylation and other gp120-specific consid-
erations necessitated modifications (Fig. 1). First, as rates of
exchange between unliganded and CD4-bound states of gp120
were expected to vary dramatically, we sought to account ex-
plicitly for the effect of complex dissociation during the ex-
change reaction. We solved the differential equation describing
gp120-CD4 complex dissociation following the 2-fold dilution
of the complex into deuterated solvent (see Materials and
Methods). Surface plasmon resonance was used to define k
, and K
of the CD4-gp120 binding reaction, and measure
ments were made at 5°C to approximate HDX conditions. At
5°C, four-domain CD4 displayed a k
[(2.65 0.09) 10
], a k
[(1.26 0.05) 10
], and a K
0.2) 10
M] to deglycosylated YU2 core gp120 that were
similar to previously published results with deglycosylated
HXBc2 gp120 at 37°C (37). Analogous measurements were
also made with glycosylated gp120 (see Table S2 in the sup-
plemental material).
Second, to provide valid estimates of error for flexibility
measurements, we devised strategies to incorporate informa-
tion from established maximum entropy methods of data anal-
ysis (34) and from overlapping peptides in the mass spectrom-
etry (see Materials and Methods; see also Fig. S2 in the
supplemental material). Rates and errors calculated by two
different incorporation strategies showed an R
of 0.99.
Third, we derived and utilized a method of normalized as-
sessment of maximum entropy (NAME), which entailed ex-
traction of rate constants from HDX curves using maximum
entropy methods (see Fig. S3 in the supplemental material)
followed by normalization with rate constants of theoretically
unstructured peptides. The normalized values (protection fac-
tors) were then processed into free energies using maximum
entropy methods and normalization with intrinsic exchange
factors (see Materials and Methods), thereby placing the re-
TABLE 1. Fragment free energies of conformational stability for
unliganded and CD4-bound gp120 with error analysis scheme 2
G SEM (kcal mol
(kcal mol
R81-N92 2.6 0.2 2.4 0.5 0.1 0.5 0.8094
N98-E106 3.6 0.1 5.9 0.0 2.3 0.1 0.0009
D107-L111 2.9 0.2 6.4 0.0 3.5 0.2 0.0023
Q114-F223 4.2 0.1 5.0 0.0 0.7 0.1 0.0220
L226-Q258 4.1 0.9 5.9 0.1 1.8 0.9 0.1943
L261-L265 3.5 0.1 5.5 0.0 2.0 0.1 0.0028
V271-T283 3.3 0.1 5.0 0.0 1.7 0.1 0.0029
Q287-L333 3.7 0.1 3.7 0.1 0.1 0.1 0.7225
T336-Q344 5.6 0.0 5.6 0.0 0.1 0.0 0.2903
L349-F383 5.5 0.0 6.9 0.2 1.4 0.2 0.0149
N386-F391 3.4 0.2 5.2 0.1 1.8 0.2 0.0078
W393-N413 2.8 0.0 3.2 0.1 0.3 0.1 0.0193
L416-M426 4.1 0.0 5.7 0.1 1.6 0.1 0.0078
E429-L453 4.9 0.3 5.9 0.3 1.0 0.5 0.1543
T455-L483 5.1 0.1 7.6 0.1 2.5 0.1 0.0011
Y486-E492 4.7 0.1 5.3 0.1 0.6 0.2 0.0676
From an unpaired Student t test
Page 3
sults on an absolute scale. Since the protection factor is the
equilibrium constant for a portion of a protein transitioning
from an ensemble of flexible, exchanging states into an ensem-
ble of rigid, nonexchanging states, the free energy associated
with it is related to local conformational stability (14, 26).
NAME-derived energies per peptic fragment showed correla-
tions (R
) with unprocessed frequencies of 0.36 to 0.68 and
0.75 to 0.77 for unliganded and CD4-bound gp120, respectively
(see Table S3 in the supplemental material). NAME has been
implemented as a Web application (http://exon.niaid.nih.gov
/HDX_NAME); it processes sequence data, amide hydrogen/
deuterium exchange, and experimental conditions (tempera-
ture and pH) into protection factors and free energies of
conformational stability.
HDX sequence coverage and spatial resolution. We chose to
study core gp120 from the YU2 isolate. HIV-1 strain YU2 is a
primary CCR5-using isolate which was cloned without cell
culture passage (33), and the crystal structure of YU2 core
complexed with CD4 had been previously determined (Protein
Data Bank accession code 1RZK) (24). Mass spectrometry of
YU2 core gp120 produced in Drosophila S2 cells suggested
substantial variability in N-linked glycosylation and identified
only a small fraction of peptide peaks. Endoglycosidases H and
D (Endo H and Endo D) cleave N-linked glycans at the pro-
tein-proximal N-acetylglucosamine, reducing the N-linked gly-
can to a single N-acetylglucosamine and a potential 1,6-fucose.
Mass spectrometry of Endo H/D-treated YU2 core after mod-
erate pepsin digestion under optimized reducing conditions
resulted in peptide identifications covering 98% of the gp120
Spatial resolution (the size of the average HDX-character-
ized fragment) is experimentally related to the size of the
pepsin-generated fragments (17), as well as to the detection
limits imposed by the mass spectrometric analysis. Pepsin di-
gestion of unliganded and CD4-bound gp120 resulted in 38
gp120 peptic fragments, which overlapped in 16 independent
regions and covered 98% of the gp120 sequence (Fig. 2; see
Table S4 in the supplemental material). All 38 fragments were
used for analysis in accordance with scheme 1, while only 16
fragments with the highest-quality data covering each of these
regions were used for scheme 2 (see Materials and Methods;
see Fig. S2 and Table S1 in the supplemental material). The
same digestion conditions for free CD4 generated 14 peptic
fragments which overlapped in 9 regions and covered 92% of
the CD4 sequence (see Table S5 in the supplemental material).
Some of the CD4 peptides had poor signal/noise in the peptic
digest of the gp120-CD4 complex, presumably related to ion
suppression in the more crowded liquid chromatography-elu-
tion profiles. Due to these effects, only 9 peptic fragments,
which overlapped in 6 regions and covered 57% of the CD4
sequence, were observed with complexed CD4. Only the high-
est-quality fragments were used for analysis using scheme 2.
Local conformational stability of unliganded gp120. Having
sorted through experimental details related to obtaining HDX
measurements on a highly glycosylated sample, we could begin
to analyze biologically relevant results, especially with regard
to the local conformational stability of unliganded gp120.
HDX measurements of unliganded, deglycosylated core gp120
showed good reproducibility, with an average standard devia-
tion of only 4% in deuterium incorporation. Meanwhile, unli-
ganded gp120 HDX measurements across all peptic fragments
ranged from 18 to 100% deuterium incorporation, with an
average variation between fragments of 31% over the time
course of exchange. Variations in HDX measurements were
thus 8-fold greater than the precision of the measurements.
In light of the observed conformational diversity of core
gp120 in various ligand-bound states (see Fig. S1 in the sup-
plemental material), we examined whether this diversity was
reflected in heightened exchange, as might be the case if un-
liganded gp120 was flexibly disordered. We observed an overall
protection factor of 2.5 10
3.3 10
for unliganded
gp120 peptic fragments covering 98% of the core. This result
indicated that unliganded gp120 exchanges at a rate 10,000-
fold lower than an unstructured peptide of the same compo-
sition, suggesting that unliganded gp120 is substantially or-
In an analysis of the diversity of core gp120, it was clear that
most of the structural variation occurs in the gp120 inner
domain/bridging sheet region, while the outer domain re-
mained essentially in the same conformation in all crystallo-
graphic studies (38). We thus examined domain-specific effects
to see if the inner-domain structural diversity was reflected by
heightened HDX. We observed that the inner domain showed
a 21-fold-more-rapid exchange than the outer domain, a sig-
nificant difference, but still 7,100-fold slower than that of an
unstructured peptide of the same composition (Fig. 3). The
results suggest that flexibility might play a role in the confor-
FIG. 1. Schematic of HDX procedure. Protein expression and
preparation were devised to minimize glycan heterogeneity while
maintaining biological integrity of the analyzed samples. Nonetheless,
heterogeneity was still observed after deglycosylation to the protein-
proximal N-acetylglucosamine (GlcNAc), necessitating modification of
standard HDX-mass spectrometry analysis to include peptic fragments
with no sugar or with GlcNAc or GlcNAc with fucose additions (Fig.
2). The results were processed by normalized assessment of maximum
entropy (NAME), a method described in the present study. These
alterations have been implemented to allow for calculation of free
energies of conformational stabilization from HDX frequencies (http:
//exon.niaid.nih.gov/HDX_NAME). Asterisks indicate steps applicable
to complexed proteins. Error analysis schemes are shown in Fig. S2 in
the supplemental material.
10314 KONG ET AL. J. V
Page 4
mational diversity of unliganded gp120. However, the results
also indicate that a substantial portion of the ensemble of
conformations that make up the inner domain/bridging sheet
appear to have considerable local conformational stability.
In terms of still finer spatial resolution, different levels of
conformational stability were observed for different regions of
unliganded gp120. Here, the free energies were mapped onto
the SIV unliganded structure since it better illustrates the
possibility of large conformational changes (Fig. 4). Two peptic
fragments (the N terminus of the inner domain as well as the
V4 loop on the outer domain) (Fig. 4A and Table 1) appeared
particularly flexible, exhibiting low G values of conforma-
tional stability and complete exchange within 15 s, more than
4 standard deviations above the average exchange rate. Fast
exchange for the N terminus (fragment 1, R81-N92) and the
V4 loop (fragment 12, W393-N413) was expected, as these
regions were mostly unstructured in the unliganded crystal
structure (6). Two particularly stable fragments were observed.
These encompassed part of the -sandwich of the inner do-
main (fragment 16, Y486-E492) and a portion of 2-helix of
the outer domain (fragment 9, T336-Q344). The hydrogen-
bonding structure inherent in -helixes may stabilize fragment
9. Fragment 16 is partially buried and intimately associated
with the N-terminal -sandwich, which is the only portion of
the inner domain that is conserved between unliganded and
CD4-bound states (6, 30), and structural analysis of a gp120
FIG. 2. HDX of HIV-1 gp120 in unliganded and CD4-bound states: peptic fragments, exchange rates, sequences, and secondary structures. The
rate of amide deuterium incorporation is related to the stability of backbone-hydrogen bonds, which in turn is reflective of the local conformational
stability of the protein. Here the gp120 sequence is shown at the top of each row (labeled “HIV1 YU2”). Residues 252 to 483 form the outer
domain of gp120, while the rest of the sequence forms the inner domain. Bars below the sequence correspond to the regions encompassed by 16
peptic fragments (explicitly numbered 1 to 16) selected for the bulk of the HDX analysis (labeled “H/D fragments”). The individual fragments are
colored in rainbow fashion from the N to the C terminus in the same way as in Fig. S1 in the supplemental material. Lines under these bars
represent overlapping peptic fragments, which were used to estimate experimental error (see Fig. S2 in the supplemental material) (labeled “peptic
fragments”). The different styles of the lines indicate differences in glycosylation, as defined by the legend in the lower right. Below these lines,
the secondary structure from the unliganded SIV gp120 crystal structure is displayed, followed by horizontal groupings of bars depicting the peptic
fragments of unliganded gp120 and their frequency of deuterium incorporation (labeled “unliganded exchange” and depicted for five time points:
15, 50, 150, 500, and 1,500 s, using a gray scale gradient to indicate exchange from 90% [white] to 10% [black] incorporation, as shown in the
lower right key). Under this, the secondary structure from the CD4-bound YU2 core gp120 crystal structure is displayed, followed by horizontal
groupings of bars depicting the peptic fragments of CD4-bound gp120 and their frequency of deuterium incorporation (labeled “CD4-bound
exchange” and depicted for the same five time points and using the same gray scale gradient as above). In general, the rate of deuterium
incorporation was higher at exposed loops (e.g., loop V4) and lower at regions with secondary structure, although when this rate was averaged over
the peptic fragments, which were 16 amino acids long on average, this correlation decreased. Overall, unliganded gp120 displayed a significantly
lower exchange rate than a theoretically unstructured protein of the same composition, and the rate of deuterium incorporation decreased upon
CD4 binding.
Page 5
with an intact gp41 interactive region suggests that this -sand-
wich is structurally conserved (38). The results suggest that at
the extremes of fast and slow exchange, HDX measurements
reflect crystallographic expectations. However, domain-spe-
cific effects were more subtle, with a far less substantial aggre-
gate difference in HDX between the outer domain (which,
crystallographically, appears conformationally stable) and the
inner domain/bridging sheet (which, crystallographically, ex-
hibit substantial conformational diversity).
Local conformational stabilities of free and gp120-bound
CD4. The extensive crystallographic structure information
available for CD4 (30, 39, 41) as well as the similarity in
structure between free and gp120-bound forms suggested that
its analysis would serve as a good comparison for the gp120
analysis. HDX measurements of free CD4 were highly repro-
ducible, with an average standard deviation of 3% in deute-
rium incorporation over all time points, which was 8-fold lower
than the average variation between different fragments.
Overall, free CD4 displayed a protection factor of 5.1
1.3 10
. This corresponds to an HDX rate 4.5
0.6-fold lower than the average exchange rate for unliganded
gp120. The highest level of fragment deuterium incorporation
(90% at 1,500 s for C16-W28 and F98-T115) was less than
that observed for the most flexible portions of unliganded
gp120 after 15 s (Table 1; see Table S5 in the supplemental
material). Meanwhile, the most stable fragments of free CD4
were comparable in exchange rate to the most stable portions
of unliganded gp120.
HDX measurements of gp120-bound CD4 could be made
for only six peptic fragments, four fewer than with free CD4,
and only five of these fragments contained duplicate observa-
tions. Nonetheless, with an average standard deviation of 4.0%
in HDX over all time points, these measurements were only
slightly less reproducible than those of unliganded gp120, and
substantially less (5.9-fold lower) than the average variation in
exchange between fragments. When CD4 bound to gp120, its
HDX slowed by 7.0 1.7-fold, with a protection factor of
1.5 10
3.6 10
. These overall numbers obscure the fact
that a statistically significant reduction in flexibility was ob-
served in only two fragments in domain 1 (P value 0.001),
FIG. 3. Global and domain-specific amide hydrogen/deuterium exchange for gp120 (left) and CD4 (right). The local conformational stability
defined by HDX can be analyzed in terms of global and domain-specific effects for both gp120 (outer and inner domains) and CD4 (domain 1,
which interacts with gp120, and domain 2, which does not). (A) gp120 domain-specific effects. The frequencies of deuterium incorporation into
gp120 peptic fragments when unliganded (red) or CD4 bound (blue) are shown as scatter plots for the outer domain (circles) and the inner domain
(triangles). Red and blue horizontal bars indicate averages and standard deviations between peptic fragments, and black horizontal bars indicate
standard deviations between experiments. Gray lines connect the same peptic fragments between unliganded and CD4-bound states. Although
CD4 interacts primarily with the outer domain, fragments from both the inner and outer domains of gp120 show considerably changes in rate of
deuterium incorporation when bound by CD4. (B) CD4 domain-specific effects. The frequencies of deuterium incorporation into CD4 peptic
fragments when free (red) or gp120 bound (blue) are shown in a scatter plot, displayed in a manner analogous to that described for panel A. In
contrast to the results in panel A, only fragments of CD4 that interact with gp120 show significant changes in rate of deuterium incorporation.
(C) Domain-specific protection factors. The average protection factors are shown in bar graphs for gp120 (left) and CD4 (right). Protection factors
are the theoretical rates of unstructured peptides divided by the experimentally obtained rates of fragment exchange. Errors were obtained using
scheme 2 (see Fig. S2 in the supplemental material).
10316 KONG ET AL. J. V
Page 6
FIG. 4. HDX-determined conformational stability as mapped onto gp120 and CD4 crystal structures. Combining HDX-determined stability
data with atomic-level structural information allows the local conformational stability of gp120 and CD4 to be visualized. (A) Representations of
local gp120 conformational stability. Energies of HIV-1 gp120 conformational stability for 16 peptic fragments (Table 1) were mapped onto a
homology model of unliganded gp120 (top row) and onto the CD4-bound crystal structure of YU2 core gp120 (second row). Structures are
displayed in C-worm representation. Peptic fragments are colored and numbered according to their positions in sequence (using the same color
scheme described in the legend to Fig. 2 and in Fig. S1 in the supplemental material), with C-worm thicknesses corresponding to energies of
conformational stability (as shown in the key on the right). Dotted lines indicate regions that were not measured. Although the conformational
diversity of unliganded gp120 suggests that it would be represented more accurately as an ensemble of structures, we nonetheless have chosen a
single structure, the unliganded SIV crystal structure, to facilitate representation. Alternative representations with gp120 shown in b12-bound and
CD4-bound conformations are shown in Fig. S6 in the supplemental material. (B) Molecular surface representations of gp120 and CD4. gp120 and
the gp120-binding site on CD4 are colored red, while CD4 and the CD4-binding site on gp120 are colored yellow. The inner and outer domains
of gp120 and domains 1 and 2 of CD4 are labeled. (C) Representations of local CD4 conformational stability. Energies of conformational stability
for nine peptic fragments of CD4 (see Table S1 in the supplemental material) are mapped onto the free structure of CD4 (left) and the
gp120-bound crystal structure from the YU2 core-CD4 complex (right). Structures and colors use a scheme analogous to that defined for panel
A. (D) Plot of change in conformational stability of fragments from gp120 (yellow) and CD4 (red) against how much of these fragments fall within
the binding interface. CD4 displays change in stability only at the interface, while gp120 displays change outside it. (E) Plot of change in
conformational stability of fragments from gp120 and CD4 against the root-mean-square deviations (RMSD) of the same fragments going from
unliganded to liganded conformations. There is no correlation between change in stability and crystal structure-implicated motions of the proteins
(see Fig. S5 in the supplemental material). Images were generated with PyMOL.
Page 7
which is the part of CD4 that contacts gp120 (fragment 3,
K29-F43; fragment 4, K46-T81) (Fig. 4B and C; see Table S5 in
the supplemental material). Thus, the overall protection factor
of domain 1 increased much more than the protection factor of
domain 2 (Fig. 3C). Also, this increase in conformational sta-
bility correlated significantly with the surface area involved in
interface contact (R
0.995, P value of 0.0004 from Pearson’s
correlation), suggesting that reduction in flexibility was iso-
lated to the site of binding (Fig. 4D).
Local conformational stability for CD4-bound gp120 and
comparison with gp120 in an unliganded state. With HDX
measurements for unliganded gp120 as well as for free and
gp120-bound CD4 providing a context, we could proceed with
an analysis of CD4-bound gp120. HDX measurements, when
corrected for complex dissociation, had an average standard
deviation of 4.4% deuterium incorporation over all time
points. This was 7.5-fold lower than the 33% average variation
between peptic fragments and similar in reproducibility to that
of unliganded gp120. Globally, a significant reduction in HDX
from unliganded gp120 was observed. The overall exchange
rate of CD4-bound gp120 was (3.7 0.1) 10
, which
was 10 1.8-fold lower than that of unliganded gp120. This
translated to a protection factor of 2.46 10
9.5 10
. The
inner-domain exchange rate was 11-fold higher than that of the
outer domain, compared to 21-fold higher when gp120 was
unliganded (Fig. 3C). CD4 binding reduced the exchange of
the inner domain by 6-fold and the exchange of the outer
domain by 3-fold.
Unlike the case with CD4, the overall reduction in deute-
rium incorporation was not limited to a small proportion of
peptic fragments. We observed reduction in HDX in 15 of 16
peptic fragments. However, reductions for only eight of these
fragments achieved statistical significance (Table 1; see Table
S1 in the supplemental material). These eight fragments were
located primarily on the face of gp120 containing the CD4-
binding site. At the binding site, fragments 7 (V271-T283), 10
(L349-F383), 11 (N386-F391), 13 (L416-M426), and 15 (T455-
L483) were significantly stabilized. Adjacent to the binding
site, 1-helix (fragments 2 [N98-E106] and 3 [D107-L111]) was
stabilized; farther from the binding site, on the face of gp120
opposite the site of CD4 binding, fragment 6 (L261-L265) was
also stabilized.
Despite a seeming visual correlation between site of CD4
binding and reduction in gp120 HDX (Fig. 4A), an R
value of
only 0.01 was observed between changes in free energies of
conformational stability and surface area involved in the bind-
ing site on gp120 (Fig. 4D). In part, this may relate to the size
of some of the peptic fragments, which may obscure reductions
in flexibility. For example, fragment 14 (E429-L453) is 35 res-
idues in length but makes contact only at residues 429 and 430.
A more biologically relevant effect may relate to the large
structural rearrangement induced in the gp120 inner domain
by CD4 binding. Notably, the largest reduction in HDX occurs
at the -helix spanned by fragment 3 (D107-L111), which does
not contact CD4. This corroborates the observation by Chen et
al. (6) that charged residues are buried during the conforma-
tional change between unliganded and CD4-bound states of
gp120 at this -helix. Ultimately, however, a comprehensive
molecular explanation is difficult, as unliganded gp120 is likely
best represented by a diverse ensemble of structures.
Conformational diversity of gp120 is a central feature of its
biological function in entry and immune evasion. In terms of
immune evasion, part of the conformational diversity relates to
the large rearrangements induced by CD4, allowing for highly
conserved surfaces that make up the coreceptor-binding sur-
face to be hidden from the immune system prior to CD4
binding at the cell surface (27, 32). Part also relates to the
decoy strategies involving the elicitation of nonneutralizing
antibodies (7). The bottom line is that gp120 in many ways
resembles the conformational machines involved in fusion,
rather than the generally more rigid receptor-binding compo-
nents (38). While the unusual nature of gp120 has been ex-
plored with methods ranging from X-ray crystallography to
titration calorimetry and glutaraldehyde fixation of antigenic
populations, none of these allow for a spatially resolved por-
trait of local conformational stability. Here we use HDX to
establish such a portrait of HIV-1 gp120, in unliganded and
CD4-bound states.
The usefulness of HDX as a probe of local conformational
stability is well established (2, 10–12, 14, 19–21, 25, 26), and a
number of different HDX methodologies have been devel-
oped. Virtually all of these use mass spectroscopy to assess the
degree of deuterium incorporation in dismantled fragments,
frozen after amide hydrogen/deuterium exchange by low pH
and then reassembled into a spatially resolved portrait of the
intact protein. We modified established methods to measure
local conformational stability (Fig. 1). Many of these modifi-
cations were previously anticipated (e.g., maximum entropy
methods, which extract the maximally unbiased probability dis-
tributions of exchange rates from the frequencies of exchange
and their standard deviations) (34, 47). We used previously
described software from S. W. Englander and colleagues to
calculate the rate constants of the peptic fragments investi-
gated by HDX as theoretically unstructured peptides (see ref-
erence 3). These rate constants were normalized by the HDX
data to obtain protection factors, which allow our observations
to be placed on an absolute scale (14, 26). The free energies
calculated from protection factors are measures of conforma-
tional stability (13). We observed that NAME-derived energies
showed significant correlation with unprocessed exchange fre-
quencies. The average R
values between NAME energies and
unprocessed frequencies were 0.50 for unliganded gp120 and
0.76 for CD4-bound gp120 (see Table S3 in the supplemental
material). When mapped onto the crystal structures of unli-
ganded and CD4-bound gp120, HDX frequencies at 50 s gen-
erated a picture of conformational stability (see Fig. S4 in the
supplemental material), extremely similar to the one produced
by the free energies (Fig. 4).
We analyzed the correlation between our HDX-determined
free energies of local conformational stability and crystal struc-
ture-determined parameters of B value, accessible surface
area, and frequencies of hydrogen bonding and -sheet/-helix
(see Fig. S5 in the supplemental material). Interestingly, the
only statistically significant correlations were between CD4-
bound gp120 and average accessible surface areas, between
CD4-bound gp120 and frequencies of -sheets/-helices, or
between gp120-bound CD4 and B value. Our results show that
the conformational stabilization of gp120 by CD4 is complex,
Page 8
involving the fixation of regions both distal and proximal to the
binding site. This is in contrast to the conformational stabili-
zation of CD4 by gp120, which is localized to the site on CD4
that binds gp120. These observations corroborate crystal struc-
tures showing large, global conformational changes in the in-
ner domain of gp120 and small, localized ones in CD4. How-
ever, our HDX data suggest that local conformational stability
does not predispose a particular region in either gp120 or CD4
to conformational changes but does correspond to larger re-
folding reactions of domains. For example, crystal structures of
SIV unliganded gp120 and HIV-1 CD4-bound gp120 show
substantial structural rearrangement in the inner domain but
little alteration of the outer domain (6, 30). Such asymmetry
was observed with HDX for both HIV-1 unliganded and CD4-
bound gp120: the outer domain was more than an order of
magnitude more stable than the inner domain (Fig. 3C). How-
ever, no statistically significant correlation was observed be-
tween local changes in structure and changes in conforma-
tional stability over the peptic fragments (Fig. 4E).
Our results indicate that unliganded gp120 is not unstable.
How then to explain the extraordinary reduction in entropy in
the gp120-CD4 binding reaction if both unliganded gp120 and
free CD4 are not unstructured? Theory indicates that reduc-
tions in entropy can come from two factors: protein folding or
solvent release. Crystallographic studies suggest that at least
part of the entropic reduction relates to the release of ordered
solvent from the surface of a highly hydrated unliganded gp120
(6, 30). However, while changes in surface burial correlated
well with changes in conformational stability for CD4 (R
0.99), no correlation was observed with gp120, suggesting that
solvent exclusion plays a general but complicated role in con-
formational stability. Likely a combination of solvent effects
and the sampling of fewer, more stable, structural conforma-
tions together explain observed entropic changes (Fig. 5).
Recently, analysis of HIV-1 gp120 with intact N and C ter-
mini (38) indicated that a “layered” gp120 architecture, with
considerable conformational diversity in the inner domain,
may account for a portion of the high entropy content of the
unliganded state. The gp120 layers are not unstructured in the
manner of a flexible loop but exist as a diverse ensemble of
conformations, each significantly different from the other and
each with significant conformational stability, all anchored to a
conformationally invariant -sandwich. Binding by CD4 re-
duces this diverse and highly entropic, though not unstruc-
tured, ensemble to a “single” conformation. In line with this,
our results show that gp120 fragments spanning the -sand-
wich (fragments 1 [R81-N92], 5 [Q114-F223], and 16 [Y486-
E492]) display much less change in conformational stability
upon CD4 binding than fragments spanning the layers (frag-
ments 2 [N98-E106], 3 [D107-L111], and 4 [Q114-F223]) (Ta-
ble 1). In particular, our data suggest that stabilization of the
-helix spanned by fragment 3 (D107-L111) is important for
the conformational convergence in the inner domain. The
present HDX analysis of HIV-1 gp120 thus provides not only
definition of local conformational flexibility but also insight
FIG. 5. Model of the transition from unliganded to CD4-bound states of gp120. HDX results combined with numerous crystal structures
suggest that the unliganded state of HIV-1 gp120 consists of a diverse ensemble of relatively stable conformations, with slow interconversion
between different, highly solvated conformations. The left side of the figure shows C-ribbon representations for polypeptides from different gp120
crystal structures, colored from the N terminus (red) to the C terminus (purple) as in Fig. 2 and 4 and Fig. S1 in the supplemental material. Blue
spheres represent solvating waters. HDX of unliganded and CD4-bound gp120 confirm the conformational stabilization of gp120 by CD4,
particularly around the surface of gp120 involved in contact with CD4. However, much of the stabilization is not within the interface and therefore
does not correlate with solvent exclusion. Also, regions in the inner domain (with no CD4 contact) appear to have been more stabilized than those
in the outer domain (which actually contact CD4). This is in line with evidence from crystal structures of gp120 bound to different ligands that show
greater conformational differences in the inner domain. The right side of the figure illustrates the changes in gp120 upon CD4 complexation. Two
features are highlighted, the change in gp120 conformational stability and the loss of solvent at the interface of gp120 with CD4. Overall, this is
consistent with a model of CD4 inducing allosteric changes that fix the conformation of one side of gp120. Surfaces on gp120 facing away from
the site of CD4 interaction are only slightly affected.
Page 9
into the thermodynamic and structural parameters governing
the CD4-gp120 binding reaction.
We thank S. W. Englander and Z. Zhang for Excel macros and
LAPLACE software used in data analysis, E. Freire, Q. Sattentau, A.
Scho¨n, L. Shapiro, and members of the Structural Biology Section,
Vaccine Research Center, for discussions or comments on the manu-
script, V. Gopalan and Y. Huyen for implementing a Web-based
version of the HDX analysis, M. Posner for antibody F105, J. Stuckey
for assistance with figures, and the NIH AIDS Research and Refer-
ence Reagent Program for CD4.
Support for this work was provided by the NIH Intramural Research
Program, a grant from the Bill and Melinda Gates Foundation Grand
Challenges in Global Heath Initiative, and the NIH/Oxford/Cam-
bridge Graduate Partnerships Program.
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    • "Although the utility of DXMS is often limited by the lengths of the proteolytic fragments that can be produced, sometimes restricting the area of investigation to less than 50% of the protein sequence, it can often provide a key insight for the design of crystallizable constructs. Furthermore, when coupled with structure, DXMS can be mapped onto the protein structure to provide a three-dimensional map of flexibility that can further guide protein-engineering efforts and mechanistic studies (Guttman et al., 2012; Kong et al., 2010; Zhang et al., 2012; Chung et al., 2011).Figure 5 Deuterium-exchange mass spectrometry (DXMS) analysis. DXMS was used to guide the construct design used for determining the crystal structure of a putative ethanolamine-utilization protein from Salmonella typhimurium. "
    [Show abstract] [Hide abstract] ABSTRACT: Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.
    Preview · Article · Feb 2016 · Acta Crystallographica Section F: Structural Biology Communications
    • "In the cryo-electron tomogram, acid-activated influenza virus is shown at early stages of membrane fusion with a synthetic liposome. HDX-MS has also provided significant new information about the initial stages of HIV Env activation upon CD4 receptor binding [2,15,52,53], which had previously been inferred at low-resolution from EM studies456109] or crystal structures of truncated subunit constructs [49,50]. HDX-MS was used to compare native-like Env trimers (SOSIP.664 "
    [Show abstract] [Hide abstract] ABSTRACT: The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.
    Preview · Article · Jan 2016 · Viruses
    • "Infection of CD4 positive cells by HIV-1 is realized via a series of protein:protein interactions that escort the virus through specific " checkpoints " which include sequential recognition events of two cellular receptors with the viral envelope (trimeric gp120 + gp41). The binding of HIV-1 gp120 to cellular CD4 is the first of these critical steps [1,2], triggering conformational rearrangements in both proteins, forming and revealing CD4 induced (CD4i) epi- topes34567. These neo-epitopes have been demonstrated by the isolation of discriminating monoclonal antibodies (mAbs) that show a distinct preference8910111213 or an absolute stringent requirement14151617 for the gp120:CD4 complex as compared to binding of either CD4 or gp120 alone. "
    [Show abstract] [Hide abstract] ABSTRACT: HIV-1 infection of target cells is mediated via the binding of the viral envelope protein, gp120, to the cell surface receptor CD4. This interaction leads to conformational rearrangements in gp120 forming or revealing CD4 induced (CD4i) epitopes which are critical for the subsequent recognition of the co-receptor required for viral entry. The CD4-bound state of gp120 has been considered a potential immunogen for HIV-1 vaccine development. Here we report on an alternative means to induce gp120 into the CD4i conformation. Combinatorial phage display peptide libraries were screened against HIV-1 gp120 and short (14aa) peptides were selected that bind the viral envelope and allosterically induce the CD4i conformation. The lead peptide was subsequently systematically optimized for higher affinity as well as more efficient inductive activity. The peptide:gp120 complex was scrutinized with a panel of neutralizing anti-gp120 monoclonal antibodies and CD4 itself, illustrating that peptide binding does not interfere with or obscure the CD4 binding site. Two surfaces of gp120 are considered targets for the development of cross neutralizing antibodies against HIV-1; the CD4 binding site and CD4i epitopes. By implementing novel peptides that allosterically induce the CD4i epitopes we have generated a viral envelope that presents both of these surfaces simultaneously.
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