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PROTEINS: Structure, Function, and Genetics Suppl. 2:28–37 (1998)
Application of Electrospray Ionization Mass
Spectrometry for Studying Human Immunodeficiency
Virus Protein Complexes
Joseph A. Loo,* Tod P. Holler, Susan K. Foltin, Patrick McConnell, Craig A. Banotai, Nicole M. Horne,
W. Tom Mueller, Tracy I. Stevenson, and David P. Mack
Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan
ABSTRACT
Mass spectrometry (MS) with
electrospray ionization (ESI) has shown utility
for studying noncovalent protein complexes,
as it offers advantages in sensitivity, speed,
and mass accuracy. The stoichiometry of the
binding partners can be easily deduced from
the molecular weight measurement. In many
examples of protein complexes, the gas phasebased measurement is consistent with the expected solution phase binding characteristics.
This quality suggests the utility of ESI-MS for
investigating solution phase molecular interactions. Complexes composed of proteins from
the human immunodeficiency virus (HIV) have
been studied using ESI-MS. Multiply charged
protein dimers from HIV integrase catalytic
core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution
pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA
1:1 stoichiometry complex was also studied by
ESI-MS. No protein-RNA complex was observed
in the absence of zinc, consistent with the role of
the zinc finger motifs for RNA binding. Proteins
Suppl. 2:28–37, 1998. r 1998 Wiley-Liss, Inc.
Key words: electrospray ionization mass spectrometry; noncovalent complexes;
protease; integrase; nucleocapsid
protein
INTRODUCTION
The development of new volatilization/ionization
methods such as electrospray ionization (ESI) has
allowed mass spectrometry (MS) to play an increasingly important role in biomedical research. Sensitive detection and accurate molecular mass determinations for a variety of macromolecules are important
advantages of MS applications. ESI has also been
used to study the solution phase complexation processes of molecules. Biological processes are typically mediated by the intramolecular and intermolecular interactions of macromolecules. Multiply
charged gas phase ions representing intact noncovar 1998 WILEY-LISS, INC.
lently bound complexes can be detected with ESIMS, as ESI is a gentle ionization method, yielding
little to no molecular fragmentation. The reports of
the globin-heme interaction of myoglobin1 and the
FKBP-FK506 receptor-ligand complex2 obtained from
aqueous solution at near neutral (or physiological)
pH were the first to suggest that complexes that
associate through specific noncovalent interactions
in solution could be detected by ESI-MS. Since these
initial reports, a variety of noncovalent binding
systems have been studied and reported, including
antibody-antigen, protein-cofactor, protein and oligonucleotide coordination with metal ions, enzymesubstrate pairings, protein subunit complexes (quaternary structure), oligonucleotide duplex (and higher
order) assemblies, and protein-DNA/RNA complexes.3
We have used ESI-MS to study protein complexes
relevant to drug discovery efforts against the human
immunodeficiency virus (HIV). Several HIV proteins
have been identified as possible targets for drug
therapies, such as protease, integrase, and nucleocapsid protein. As part of their function, many of these
HIV proteins interact with other molecules. This
report will present several examples of protein interactions we have studied by ESI-MS, such as metal
ion binding, inhibitor binding, protein quaternary
structures, and protein-RNA interactions, as part of
our efforts in HIV drug discovery. Because of the
accuracy of the mass measurements, stoichiometry
of the binding partners is readily determined, even
for small molecule binding (e.g., inhibitors and drug
molecules) to larger molecular mass targets.
MATERIALS AND METHODS
Integrase Catalytic Core (F185K) Expression
and Purification
The synthetic HIV-1 integrase gene was shuttled
between pKK223–3 and the T7 expression vector
pET21a (Novagen, Madison, WI), using the unique
NdeI and HindIII sites, to produce the plasmid
pET21a-NY5IN. This plasmid was used to transform
Escherichia coli strain BL21(DE3). BL21(DE3) con*Correspondence to: Joseph A. Loo, Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105.
E-mail: Joseph.Loo@wl.com
Received 7 April 1998; Accepted 14 July 1998
ESI MASS SPECTROMETRY OF HIV PROTEIN COMPLEXES
taining pET21a-NY5IN were grown at 37° C with
vigorous stirring and aeration in a 2 L Omniculture
fermentor (Virtis Company, Gardiner, NY) containing superbroth (Digene, Silver Spring, MD) and 100
µg/mL ampicillin. When the optical density reached
8 (600 nm), integrase expression was induced by
addition of IPTG to a final concentration of 1 mM.
Approximately 90 minutes later, the cells were harvested by centrifugation at 5,000 x g for 20 minutes,
frozen in liquid nitrogen, and stored at ⫺80° C.
Purification, carried out at 4° C, was begun by
suspending the cells in 5 mL of lysis buffer (50 mM
HEPES, pH 7.5, 5 mM DTT, 1 mM DTPA, and 1
µg/mL leupeptin and pepstatin A) per gram of cell
paste. The suspension was passed twice through a
French pressure cell at 12,000–14,000 psi and spun
at 12,000 x g for 30 minutes. The supernatant was
discarded, and the pellet was resuspended in buffer
A (25 mM HEPES, pH 7.5, 1 mM DTPA, 1 mM DTT, 1
µg/mL each leupeptin and pepstatin A), homogenized with a Dounce homogenizer, and re-pelleted.
This second pellet was resuspended in buffer B (25
mM HEPES, pH 7.5, 10 mM CHAPS, 1 mM DTT, 1
mM DTPA, 1 µg/mL each leupeptin and pepstatin A)
containing 1 M NaCl and stirred for 1 hour to extract
the integrase. The suspension was clarified by centrifugation at 80,000 x g for 1 hour. The supernatant
was slowly diluted ten-fold with buffer B containing
150 mM NaCl, mixed with DEAE Sephacel (Pharmacia, Uppsala, Sweden), stirred on ice for 30 minutes,
and filtered to remove the resin. Heparin sepharose
(Pharmacia) was added to the filtrate, stirred on ice
for 10 minutes, and then packed into a column.
Integrase was eluted with a gradient of 250–1,000
mM NaCl in buffer B. Integrase-containing fractions
were pooled, dialyzed overnight (25 mM HEPES, pH
7.5; 10 mM CHAPS, 10 mM DTT; 500 mM NaCl and
20% glycerol), aliquoted, and stored at ⫺80° C.
Integrase concentration was determined by quantitative amino acid analysis.
Solutions for ESI-MS measurements were desalted and concentrated by centrifugal ultrafiltration. Intregrase solutions were washed with two 400
µL volumes of 5% (v/v) acetic acid, followed by two
400 µL volumes of 10 mM ammonium acetate (pH
6.5) and passing the solutions through 5,000 Da
cut-off centrifugal filtration cartridges (Ultrafree-MC with low-binding regenerated cellulose membrane; Millipore, Bedford, MA). The protein solutions were concentrated to 0.2–0.5 µg/µL.
Protease Expression and Purification
Cell strain
The E. coli strain expressing the HIV protease
NY5 was a gift from Dr. Sergei Gulnik (SAIC Frederick, Frederick, MD). The host cell line was BL21(DE3)
and the vector was pET24.
29
Expression
The culture was started by inoculating a shake
flask containing 200 mL of Luria Broth (Becton
Dickinson, Cockeysville, MD) plus 100 µg/mL ampicillin with 1 mL of a 20% glycerol stock. The shake
flask was grown at 30° C. After the shake flask
became cloudy (8 hours), 1 mL of the shake flask
contents was used to inoculate a 10 L fermentor. The
media for the fermentation consisted of 2% (w/v) of
the following: yeast extract (Difco, Detroit, MI);
acidicase peptone (Becton Dickinson); casitone
(Difco); gelatone (Difco); 0.2% (w/v) of the following: KH2PO4 anhydrous, K2HPO4 anhydrous,
Na2HPO4●7H2O; and 100 µg/mL ampicillin. The
culture was maintained at pH 6.8 with lactic acid.
Air was introduced into the fermentor at 8 L/min,
and the temperature was maintained at 30° C. The
culture was grown in the fermentor to an optical
density of 10 at 600 nm, the temperature was raised
to 37° C, and recombinant expression was induced by
addition of isopropyl ␤-D-thiogalactopyranoside
(IPTG) to a concentration of 3.5 mM. The fermentor
was harvested 3 hours after induction by using
hollow-fiber microfiltration and centrifugation. The
cell paste was stored at ⫺70° C.
Cell disruption
Cell lysis was carried out by using a Dyno-Mill
KDL (Glen Mills, Clifton, NJ) with a 600-mL chamber containing 500 mL of 0.25–0.5 mm lead-free
glass beads. The lysis solution was 50 mM sodium
phosphate buffer, pH 7.2, containing 1.0% Triton
X-100, 10 mM MgCl2, and 0.045 mL of Benzonase
(American International Chemicals, Natick, MA)
per liter. The cell paste (350–400 g) was resuspended
to 1.2 L with lysis solution and run through the
Dyno-Mill twice at 100 mL/min. The jacket temperature was ⫺13° C and the agitator speed was 4,200
rev/min. The suspension was then centrifuged for 90
minutes at 4° C in a DuPont Sorvall (Wilmington,
DE) centrifuge using a GS-3 rotor at 9,000 rev/min.
Inclusion body isolation
The inclusion body isolation was based on the
procedures of Estape and Rinas4 and Marston et al.5
The pellet from the centrifugation was thoroughly
resuspended using a Polytron PT 3000 homogenizer
equipped with a PT DA 3012/ S generator (Brinkmann Instruments, Westbury, NY) at 10,000 rev/min
with 750 mL of 50 mM potassium phosphate buffer,
pH 8.0, containing 10 mM EDTA, 0.06 mL/L Benzonase, and 2,500 U/mL lysozyme from chicken egg
white (Sigma Chemical, St. Louis, MO). The suspension was adjusted to pH 7.5 with NaOH or HCl and
stirred for 2 hours at 37° C. Glycine was then added
to a concentration of 50 mM and the pH was adjusted
to 10 with NaOH. Triton X-100 was added to a
concentration of 1%, DTT was added to a concentra-
30
J.A. LOO ET AL.
tion of 10 mM, and the suspension was stirred for 18
hours at 4° C. Water (750 mL) was added, and the
suspension was centrifuged as described above. The
pellet was resuspended as described above with 1.5 L
of water; the suspension was stirred for 30 minutes
and centrifuged as described above. The water wash
step was repeated once more. The suspension was
resuspended with 1.5 L of 95% ethanol, stirred for 1
hour, and centrifuged as described above. The pellet
was lyophilized in a Dura-Dry µP Freeze Dryer (FTS
Systems, Stone Ridge, NY) at 10 mT for 18 hours and
stored at ⫺70° C.
the enzyme solution was diafiltered versus 2 L of 50
mM sodium acetate buffer, pH 5.5, concentrated to
7–10 mg/mL with an Amicon-Millipore YM 10 membrane at 5° C and stored at ⫺70° C.
Protease solutions for ESI-MS analysis were desalted by using 5 kDa cutoff centrifugal ultrafiltration cartridges (Millipore). The 7–10 mg/mL protease samples were washed with several volumes of a
solution containing 0.1 mM pepstatin A inhibitor
(Sigma), 0.3 mM DTT, and 10 mM ammonium acetate (pH 5). The addition of pepstatin A prevented
degradation of the enzyme.
S-Sepharose chromatography
Activity assay
All steps were carried out at room temperature.
The dried pellet (6–9 g) was extracted by resuspending in 330 mL of 50 mM sodium borate buffer, pH 8.6,
containing 8 M urea, 2 mM EDTA, and 10 mM DTT
(solvent A) using the Polytron homogenizer and
centrifuged as described above. The extraction of the
pellet was repeated twice. The supernatants from
the three extractions were combined and Triton
X-100 was added to a concentration of 1%. The
solution was adjusted to pH 8.6 with NaOH and
applied to a S-Sepharose Fast Flow column (9 ⫻ 10
cm; Pharmacia) equilibrated with solvent A. The
column was washed with solvent A at 200 mL/min
until A280nm of the column effluent was zero. The
enzyme was eluted with a linear salt gradient (solvent B: 0.5 M NaCl in solvent A, total gradient
volume of 4 L) at 200 mL/min. Fractions containing the purified, denatured enzyme as determined by SDS-PAGE were combined and stored at
⫺70° C. Matrix-assisted laser desorption/ionization
(MALDI)-MS (Voyager Elite-DE time-of-flight mass
spectrometer, PerSeptive Vestec, Framingham, MA)
was used to verify the molecular mass of the protein.
The activity of the enzyme was measured by the
hydrolysis of the substrate His-Lys-Ala-Arg-Val-Leup-nitro-Phe-Glu-Ala-Nle-Ser-NH2 (BACHEM, King
of Prussia, PA)7,8 in 72 mM sodium acetate buffer, pH
4.7, containing 0.9 mM EDTA, 0.14 M NaCl, 0.09%
(w/v) PEG 8000, and 0.9 mM DTT. Samples of
enzyme (40 nM) were incubated with substrate
(25–400 µm) at 37° C for 10 minutes, and the
reaction was quenched by the addition of 1.67% (v/v)
trifluoroacetic acid (TFA). Fifty microliters of each
quenched reaction mixture was separated on a
DYNAMAX C18, 5 µm column (4.6 ⫻ 50 mm)
equipped with a DYNAMAX C18, 5 µm guard column (4.6 ⫻ 15 mm) (RAININ Instruments, Woburn,
MA). Unconverted substrate and hydrolysis products were resolved in a linear acetonitrile gradient
(10% solvent B to 40% solvent B in 4 minutes;
solvent A: 0.1% TFA in water, solvent B: 0.07% TFA
in acetonitrile) and detected by monitoring at 220
nm. Chromatography was performed at 1.5 mL/min
at room temperature. Kinetic analysis of the data
generated a KM ⫽ 23 µM and kcat of 0.64 sec-1.
Enzyme refolding
Nucleocapsid Protein-RNA Sample
Preparation
Purified, denatured enzyme (225 mg) was diluted
to 0.16 mg/mL with 8 M urea, and DTT was added to
a concentration of 10 mM. The enzyme concentration
was determined at 280 nm, using an A280nm of 1.17
for a 1 mg/mL solution, calculated from the amino
acid composition (modification of the procedure of
Gill and Von Hippel6) and an average molecular
mass of 10,779 Da. The solution was diafiltered
versus 2.4 L of 25 mM sodium phosphate buffer, pH
7.0, containing 25 mM NaCl, 10 mM DTT, and 10%
glycerol with an Amicon-Millipore (Bedford, MA)
S1Y10 spiral cartridge. The temperature of the
enzyme solution was maintained at 0–5° C by the
use of a heat exchanger coupled to a refrigerated
recirculator. The enzyme solution was concentrated
to 200 mL in the spiral cartridge and diafiltered as
described above versus an additional 400 mL of 25
mM sodium phosphate buffer, pH 7.0, containing 25
mM NaCl, 10 mM DTT, and 10% glycerol. Finally,
The expression and purification of nucleocapsid
protein (NCp7) was described previously.9 Peptides
corresponding to the zinc finger regions of NCp7
were synthesized by the University of Michigan
Protein and Carbohydrate Structure Facility (Ann
Arbor, MI).
RNA samples were chemically synthesized using
phosphoramidite chemistry, purified by polyacrylamide gel electrophoresis, and characterized by enzymatic sequencing and ESI-MS. Gel-purified RNA
samples for ESI-MS binding studies were further
desalted by cold ethanol precipitation as ammonium
acetate salts and lyophilized.10,11 To assure that the
RNA had the proper secondary and tertiary structure, the 10 µM solutions in 10 mM ammonium
acetate, pH 6.9, were annealed by heating for 4
minutes at 95° C and cooled slowly (3 hours). Methanol (10% v/v) was added to the protein-RNA solution
before ESI-MS experiments to enhance the stability
ESI MASS SPECTROMETRY OF HIV PROTEIN COMPLEXES
31
Fig. 1. ESI mass spectra (plotted on the
same m/z axis) of HIV integrase catalytic
core (F185K) showing only the (a) 18 kDa
monomer species detected from a pH 2.5
solution (2:1 v/v acetonitrile:water with 3%
acetic acid) and (b) the 36 kDa dimer protein
from a pH 6.5 solution (10 mM ammonium
acetate). The glass capillary ESI interface
was used to acquire the spectra.
of the ESI-MS signal without altering the resulting
mass spectra.
Mass Spectrometry
ESI-MS was performed with a double focusing
hybrid mass spectrometer (EBqQ geometry, Finnigan MAT 900Q, Bremen, Germany) with a mass-tocharge (m/z) range of 10,000 at 5 kV full acceleration potential.12 A position and time-resolved ion
counting (PATRIC) scanning array detector with an
8% m/z range of the m/z centered on the detector
was used.13,14
For some of these experiments, an ESI interface
based on a heated glass capillary inlet was used.15
This interface uses a countercurrent flow of warm
nitrogen gas (⬃60° C) to aid the droplet and ion
desolvation process. For the detection of noncovalently bound gas phase complexes, it is important to
have fine control of parameters affecting the desolvation process. The energy of gas phase collisions in the
ESI interface region is controlled by the voltage
difference between the tube lens at the metalized
exit of the glass capillary and the first skimmer
element (⌬ VTS) and is used to augment the desolvation of the ESI-produced droplets and ions. Additionally, spraying the aqueous solutions ‘‘off axis’’ relative to the capillary inlet produces mass spectra with
minimal solvation of the ions and with an increase in
sensitivity. Spraying ‘‘on axis’’ relative to the capillary inlet produces broad peaks, as larger droplets
are sampled by the MS interface. A stream of SF6
coaxial to the spray suppressed corona discharges,
especially important for ESI of aqueous solutions.
Solution flow rates delivered to the ESI source were
in the 0.5–1.0 µL min-1 range.
Other experiments used a heated metal capillary
inlet16 with a low flow micro-ESI source.11 Control of
the analyte flow rate from 50–200 nL min-1 was
accomplished by pneumatic pressure. Capillary temperatures were maintained around 150–200° C for
noncovalent complex studies and 210–225°C for other
work. A countercurrent gas flow was not necessary
for desolvation. However, as for the glass capillary
inlet, the voltage difference between the tube lens
and the skimmer lens (⌬ VTS) was critical to control
the energy of collisions in the interface region.
RESULTS AND DISCUSSION
HIV Integrase: Protein Dimerization
In addition to other viral and host cell proteins,
HIV infection requires the activity of the three
enzymes encoded by the viral pol gene: protease
(PR), reverse transcriptase (RT), and integrase (IN).
Integration of a double-stranded DNA copy of the
retroviral genome into the host chromosome is essential for viral replication. To facilitate this process, IN
catalyzes a coordinated series of DNA cutting and
joining reactions. RT and PR are the targets of
several HIV inhibitors currently used in clinical
practice. However, HIV integrase protein may potentially be a new target for anti-AIDS drugs.17
The crystal structure of the catalytic core of HIV-1
integrase was solved by Dyda et al.18 The limited
solubility of the protein hindered its detailed characterization. However, crystallization was made possible by the discovery of a mutation, F185K, which
enhances solubility but does not affect the biochemical properties of integrase. Integrase functions minimally as a dimer, consistent with the crystal structure.18–20
The ESI mass spectrum of IN F185K catalytic core
(residues 50–212, Mr 18171) from denaturing solution conditions shows a pattern of multiply charged
ions of relatively high charge consistent for the 18
kDa monomeric species (Fig. 1; see Table I for
molecular weight measurements). From an aqueous
solution at pH 6.5, only three multiply charged ions
of relatively low charge (11⫹–13⫹) and at relatively
high mass-to-charge (m/z ⬎ 3,000) from m/z 3,000–
32
J.A. LOO ET AL.
TABLE I. ESI-MS Molecular Weight Measurements
HIV integrase
Monomer†
Dimer
HIV protease
Monomer†
Dimer
Dimer ⫹ pepstatin A
HIV NCp7
Apo-protein†
Holo-protein (⫹2 Zn2⫹)
SL3 ␺-RNA‡
Holo-NCp7 ⫹ SL3 ␺-RNA
N-terminal Zn-finger peptide
Apo-peptide†
Holo-peptide (⫹1 Zn2⫹)
C-terminal Zn-finger peptide
Apo-peptide†
Holo-peptide (⫹1 Zn2⫹)
Apo-peptide (mutant)†
Holo-peptide (mutant, ⫹1
Zn2⫹)
Mr
(sequence)
Mr
(measured)
18170.8
36341.6
18172.0 ⫾ 0.4
36345.9 ⫾ 4.7
10778.8
21557.6
22243.5
10780.0 ⫾ 0.6
21555.5 ⫾ 1.8
22242.0 ⫾ 2.8
6369.5
6496.2
4524.8
11021.0
6369.7 ⫾ 0.3
6496.6 ⫾ 0.4
4525.0 ⫾ 0.5
11023.0 ⫾ 0.5
1978.4
2041.7
1977.7 ⫾ 0.2
2041.3 ⫾ 0.3
2068.4
2131.8
2068.4
2068.0 ⫾ 0.1
2131.4 ⫾ 0.3
2067.9 ⫾ 0.2
2131.8
2131.1 ⫾ 0.2
†ESI-MS
measurements from acetonitrile/water with 2–5%
(v/v) acetic acid solutions.
‡Negative ion ESI-MS measurement from acetonitrile/water
with 0.8% (v/v) ammonium hydroxide solution.
3,700 representing the 36 kDa dimeric species are
observed. The production of a limited number of
charge state ions is believed to be related to the
protein’s native, compact structure.21 Higher protein
concentrations do not result in observation of higher
order aggregates in the mass spectrum. Increasing
⌬VTS of the atmosphere/vacuum interface from ⫹50 V
to ⫹140 V promotes dissociation of the gas phase
dimer to the monomer. Further increase of ⌬VTS
results in dissociation of covalent polypeptide backbone bonds that provide confirmation of the primary
structure (data not shown). Fragment ions of yn-type
localized near Pro amino acids are preferentially
formed. The preference for bond dissociation near
proline residues was discussed by Loo et al.22 By
carefully controlling instrumental parameters necessary for ion desolvation and increasing solution pH
to near physiological conditions, ions for the specific
noncovalently bound IN catalytic core dimer can be
detected by ESI-MS.
HIV Protease: Dimerization and
Inhibitor Binding
HIV-1 protease is a member of the aspartyl proteinase family of enzymes, including pepsin, renin, chymosin, and cathepsin D.23 PR catalyzes specific
cleavage of the viral gag and gag-pol translation
products to yield the viral enzymes and structural
proteins required for assembly of the virions. It is a
homodimeric protein made up of two 99-residue
polypeptide chains. Enzymatic activity of HIV-1 PR
maximizes near pH 6.24 Dissociation of the active
dimeric form of the enzyme results in complete loss
of activity. The stability constant, KD, of the HIV-1
PR dimer is strongly pH dependent and is 0.75 nM at
pH 5.5 and 48 nM at pH 7.5 (30° C).25
Pepstatin A (isovaleryl-Val-Val-Sta-Ala-Sta, Mr
686) is a specific inhibitor of the aspartyl proteinases.26,27 Binding of the inhibitor is associated with
significant conformational changes in the enzyme.28,29
The active site of HIV PR is composed of equal
contributions from each of the subunits. Interactions
between the inhibitor and PR involve both hydrogen
bonding and hydrophobic forces with residues in the
body of the protein and with residues in the flap
regions.
Binding of pepstatin A to HIV-1 protease as a
function of pH was investigated by ESI-MS. Pepstatin A was added to an aqueous solution containing
PR; the excess inhibitor was removed by centrifugal
ultrafiltration. Using the heated metal capillary
interface and the low-flow micro-ESI source, MS of
PR (monomer Mr 10779) in acetonitrile/water and
pH 2.5 (2.5% v/v acetic acid) yields only ions for the
monomeric protein (Fig. 2; Table I). Aliquots of the
stock PR-inhibitor solution were diluted into ammonium acetate solutions at various pH values (adjusted by addition of acetic acid or ammonium hydroxide) and analyzed by MS. ESI mass spectra acquired
from pH 4.7–6.9 solutions show ions for the ternary
PR dimer-inhibitor complex as well as uncomplexed
PR dimer and monomer (Fig. 3). However, the relative abundance of PR dimer-inhibitor complex was
higher at pH less than 6 (Fig. 3a,b) compared with
the pH 6.9 spectrum (Fig. 3c). Moreover, ESI-MS of
the PR-inhibitor solution at pH 7.4 shows only ions
for the PR monomer (Fig. 3d), i.e., evidence for PR
dimerization or complexation with pepstatin A was
not observed. These results are generally consistent
with the solution stability and enzymatic activity of
PR dimer. Maximum stability of the dimer and
ternary complexes are approximately at pH 5.5–6.0.
The HIV PR-pepstatin A ESI-MS results are similar to a previous report published by Baca and
Kent.30 The stability of the PR-pepstatin A gas phase
complex is highly sensitive to the ESI atmosphere/
vacuum interface conditions. The relative abundance of the ternary complex maximized with low
⌬VTS (⬍10 V) and low metal capillary temperatures
(Tcap ⬍ 175° C). Raising ⌬ VTS and/or Tcap results in
dissociation of the PR dimer and PR dimer-pepstatin
A complexes to the PR monomer. For example, with
⌬VTS ⫽ 0 V and Tcap set to 200° C, a significant
amount of the PR dimer complexes dissociated to the
monomeric species.
An important internal water molecule (water-301)
observed in nearly every crystal structure of the
PR-inhibitor complex27,28,31,32 was not observed in
our study nor in Baca and Kent’s report.30 Although
this water molecule is highly coordinated to the PR
structure, bridging the flaps of the enzyme to the
ESI MASS SPECTROMETRY OF HIV PROTEIN COMPLEXES
33
Fig. 2. ESI mass spectrum of HIV-1
protease/pepstatin A solution (10 µM concentration) under denaturing conditions (2:1 v/v
acetonitrile:water with 2.5% acetic acid, pH
2.5). The inset shows isotopic resolution of
the (M⫹12H)12⫹ ion. The heated metal capillary interface (Tcap 200° C) and low-flow
micro-ESI source (150 nL/min) were used.
Fig. 3. Mass spectra of the HIV-1 protease/
pepstatin A mixture acquired from 10 mM ammonium acetate solutions (pH adjusted by addition
of acetic acid or ammonium hydroxide). The
heated metal capillary temperature was 150° C.
inhibitor, the residence time is estimated to be much
shorter relative to PR dimerization and inhibitor
binding. Water molecules bound to proteins have
high mobility and are in rapid motion and exchange
in solution, even those that appear to be fixed in a
crystal.33 The half-life of dimer dissociation is approximately 30 minutes and inhibitor binding around 1
second,25 whereas the interaction of water with PR
has been estimated to be 10 nsec.32 Such kinetic
parameters may conspire to preclude the observation of water molecule binding to the gas phase
complex by MS. Water clusters or adducts to polypeptides have been observed by ESI-MS.34–36 However,
whether these water molecules are structurally relevant to the solution structure is a question that
remains to be answered. In general, the loss or
34
J.A. LOO ET AL.
Fig. 4. Amino acid sequences of nucleocapsid protein (NCp7) and the synthetic
peptides corresponding to the N-terminal
and C-terminal zinc fingers used for this
study.
Fig. 5. Deconvoluted (to the mass domain) ESI mass spectra of nucleocapsid
protein (NCp7, 20 µM concentration, 10 mM
ammonium acetate, pH 6.9, analyte flow
rate 150 nL/min) in the (a) absence and (b)
presence of 10 times excess zinc ion (Tcap
150° C). A maximum zinc stoichiometry of
two is measured in the presence of excess
zinc. The insets show the isotopically resolved deconvoluted mass spectra.
dissociation of water from the gas phase complex is
observed by MS. Recent results from Chung et al.37
suggest that for selected protein complexes, water
molecules important for the solution phase structure
may be retained in the gas phase.
HIV Nucleocapsid Protein: Zinc and RNA
Binding
Zinc finger proteins contain Cys and His residues
that coordinate zinc and form structural motifs for
interacting with nucleic acids, as displayed by several transcription factors that include zinc finger
structures for DNA recognition. HIV nucleocapsid
protein NCp7 contains two zinc fingers (or knuckles)
of type Cys-X2-Cys-X4-His-X4-Cys (where X is a
variable amino acid) that are involved in encapsulation of genomic RNA and viral assembly (Fig. 4).38,39
We40 and others41–43 have used ESI-MS to determine
the zinc-binding stoichiometry for NCp7 (2 mol of
zinc per mol of protein). An example is shown in
Figure 5. Two protons are lost for each zinc ion
complexed for CCHC-type zinc finger structures.44
The time course of ejection of zinc from NCp7 caused
by the covalent binding of various inhibitors can be
monitored by MS.40,42,43
The relative zinc-binding affinity of peptides corresponding to each knuckle can be investigated as
well.45 For example, an ESI mass spectrum from a
solution containing an equimolar concentration of
peptides corresponding to the N-terminal and C-terminal knuckles in the presence of zinc is shown in
Figure 6. Each peptide binds a maximum of one zinc
ion. However, a competitive binding experiment in
which the C-terminal knuckle peptide (Lys-Gly-Cys-
ESI MASS SPECTROMETRY OF HIV PROTEIN COMPLEXES
35
Fig. 6. Deconvoluted (to the mass domain) ESI mass spectra from an equimolar
mixture (50 µM each) of the N-terminal zinc
finger peptide and the (top) C-terminal zinc
finger peptide and the (bottom) mutant
C-terminal peptide (pH 6.9, 10 mM ammonium acetate) in the presence of zinc. The
competitive binding experiment shows
greatly reduced zinc affinity for the mutant
peptide, as the major species observed for
the mutant peptide is the 2,068 Da apoform.
Fig. 7. ESI mass spectra from a solution
containing an equimolar concentration of
NCp7 and SL3 ␺-RNA (10 µM each, 10 mM
ammonium acetate, pH 6.9) in the (a) absence and (b) presence of excess zinc ion.
Specific formation of the 1:1 stoichiometry
protein/RNA complex (where the protein is
bound to 2 mol of zinc; peaks labeled with
filled circles) is observed in the presence of
zinc. Instrumental conditions used were the
same as described for Figure 5.
Trp-Lys-Cys-Gly-Lys-Glu-Gly-His-Gln-Met-Lys-AspCys-Thr-Glu) is replaced with a mutant peptide
where the Cys6-Gly7 stretch is reversed to Gly6-Cys7
shows a greatly reduced zinc-binding affinity of the
mutant C-terminal peptide relative to that of the
N-terminal peptide (Fig. 6, bottom). Most of the
36
J.A. LOO ET AL.
mutant peptide appears as the apo-form under these
competitive binding conditions. The sequence and
residue spacings are very important for zinc coordination. Experiments performed with only the mutant peptide in the presence of excess zinc show a
maximum zinc stoichiometry of one (data not shown;
Table I).
Located between the 58 long terminal repeat (LTR)
and the gag initiation codon is the RNA sequence
referred to as the Psi (␺) site, important for encapsidation. The N-terminal zinc finger element is critical
for nucleocapsid binding to ␺-RNA. The structure of
␺-RNA contains four double-stranded stem/singlestranded loop (or stem-loop [SL]) elements.46 Three
of the four stem-loops show relatively high affinity to
NCp7; however, SL3 has been proposed as a particularly important region of ␺-RNA. De Guzman et al.47
recently reported the NMR structure of the NCp7SL3 complex. A 1:1 protein-RNA complex with a
dissociation constant of ⬃100 nM was observed.
The positive ion ESI mass spectrum of an
equimolar solution of NCp7 and an RNA consisting
of the sequence for SL3 ␺-RNA (CUAGCGGAGGCUAG, Mr 4525) is shown in Figure 7. Without any
zinc added to the solution, the most abundant ions
observed represent the apo-form of NCp7 and SL3
RNA. We had previously reported the observation of
multiply charged positive ions from RNA and
DNA.11,48 Adding zinc to the solution results in the
formation of ions for the 1:1 stoichiometry NCp7-Zn2/
SL3 RNA complex (Fig. 7b). Zinc induces structural
changes and stabilization in the NCp7 molecule, i.e.,
the zinc finger structures must be fully functional for
RNA binding. Such conformational ordering is a
prerequisite for RNA binding. A similar ESI-MS
experiment was demonstrated by Nemirovskiy et
al.,49 in which calcium-binding was required for the
EF-hand calcium-binding protein, calmodulin, to
bind specifically to the peptide melittin.
Unlike the integrase and protease dimers and the
PR dimer-pepstatin A ternary complex, the gas phase
nucleocapsid protein-RNA complex is extremely
stable. The gas phase protein-RNA complex remains
intact at elevated capillary temperatures (Tcap to
250° C) and ⌬ VTS values. The relative stabilities of
noncovalent complexes held together largely by electrostatic forces, such as positively charged proteins
and anionic oligonucleotides, was recently discussed.3,11 Electrostatic forces may be strengthened
in the absence of solvent (i.e., in vacuo), whereas
complexes held together by hydrophobic interactions
are extremely labile in the gas phase.
CONCLUSIONS
ESI-MS can be used to study protein complexes
important for HIV replication. It is important to
understand the structure and biological function of a
number of key macromolecules to choose targets for
drug discovery and to design potential inhibitors for
AIDS therapies. In the examples described in this
article and many other published accounts,3 the data
from ESI-MS experiments of noncovalent complexes
are consistent with known solution phase characteristics. It is this feature that provides the driving
force for applying ESI-MS to the study of noncovalent complexes. Whether the gas phase complex has
a similar or related structure to its solution phase
counterpart is a matter of debate.50 In some isolated
examples, the gas phase and solution phase structures share similar characteristics.51 Even if these
cases were the exception, however, MS is extremely
useful for biochemical research as a bioanalytical
tool for measuring binding stoichiometry and complex dynamics in a rapid, accurate, and sensitive
manner.
ACKNOWLEDGMENTS
The authors thank Margaret Whitton for N-terminal sequencing, Patricia J. Harvey for assistance
in the expression and isolation of NCp7 protein, and
John Domagala and Michael Reily for many helpful
discussions.
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