(Received for publication, April 10, 1997)
From the Laboratory of Structural Biology, NIEHS,
Research Triangle Park, North Carolina 27709 and the
§ Protein Expression Laboratory, NIAMS, National Institutes
of Health, Bethesda, Maryland 20892
Kinetic measurements on a fluorescent peptide
analog of the p17/p24 cleavage site of the Gag polyprotein demonstrate
the conformational selectivity of human immunodeficiency virus, type 1 protease for the trans conformation of the Tyr-Pro bond. A
mean cis/trans ratio of 0.3, and a cis trans isomerization rate constant of 0.022 s
1
are determined at T = 22 °C. This rate is in
excellent agreement with that predicted by 19F NMR studies
of structurally analogous peptides containing a fluorine/hydroxyl
substitution on the tyrosyl residue. Addition of recombinant human
cyclophilin resulted in a significant enhancement of this rate, and it
is proposed that this enzyme, which has been shown to be associated
with the Gag protein, functions as an auxiliary enzyme for the protease
during cleavage in the virion.
Three of the eight consensus sequences cleaved by HIV-11 protease involve Araa-Pro bonds, where Araa corresponds to the aromatic amino acids tyrosine or phenylalanine (1). Since imide bonds are known to exhibit conformational cis-trans heterogeneity, the existence of such cleavage sites leads to questions concerning the conformational specificity of the protease. It has been demonstrated that cleavage of Xaa-Pro imide bonds by prolidase, aminopeptidase P, and carboxypeptidase P is specific for the trans conformation of Xaa-Pro dipeptides (2-5) and further that proteases can be conformationally selective even for nearby, non-scissile peptide bonds (6-9). Indeed, this conformational specificity of chymotryptic cleavage for the P2-P3 bond has provided the basis for assays used to demonstrate the existence of peptidyl proline isomerases (10). Several lines of evidence suggest that HIV protease might be specific for the trans imide bond conformation: 1) crystallographic studies of inhibitors containing proline (11) or thiazolidine (12) indicate a trans conformation; 2) the ability of the protease to cleave ordinary amide bonds as well as imide bonds suggests a capability for cleaving trans imide bonds (1). It is important to emphasize, however, that neither of these observations provides unambiguous proof. For example, it is well established that inhibitors that exhibit close structural relationships to natural substrates can nevertheless bind to enzymes in dramatically different conformations (13).
There are several motivations for understanding the conformational selectivity of HIV protease. 1) Many of the test peptides used to assay the protease contain imide Xaa-Pro bonds. In the event of conformational selectivity, the overall kinetic characterization of these assays should be generalized to include a combination of cleavage and isomerization rates, with the specific mix determined by the physical conditions such as temperature, buffer, etc. 2) Conformational selectivity by the protease could have significant implications for the kinetics of the cleavage process in vivo, closely analogous to the effect of cis-trans imide bond isomerism on protein folding and unfolding. The recent discovery of a close association between the peptidyl proline isomerase cyclophilin and the Gag polypeptide of HIV-1 (14-16) underlines the potential significance of this proteolytic selectivity.
The kinetic behavior of a trans-selective protease considered to cleave a trans bond irreversibly has been discussed by Lin and Brandts (2) and can be described by the following kinetic scheme,
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The fluorescent peptide substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)-Arg, was obtained from Molecular Probes (Eugene, OR). The fluorinated peptide, Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln (FPhe = 4-fluoro-L-phenylalanine) was obtained from Macromolecular Resources (Ft. Collins, CO). HIV protease was obtained from Bachem, Inc. Recombinant human cyclophilin was obtained from Sigma.
MethodsHIV protease activity was measured using a Fluoroskan Ascent plate reader (LabSystems, Needham Heights, MA). Excitation and emission wavelengths were 355 and 485 nm, respectively. Assay conditions were 1.0 M NaCl, 0.2% polyethylene glycol, and 0.1 M MES, pH 5.5, at room temperature (22 °C) in 200 µl total volume. For the studies shown, data were obtained every 0.5 s for 3 min.
Fluorine-19 NMR spectra were obtained on a Varian UnityPlus 500 NMR spectrometer operating at a 19F resonance frequency of 470.996 MHz using a 5-mm 19F{1H} probe. cis/trans isomerization rates at higher temperatures were determined using magnetization transfer techniques analogous to those described previously in 19F NMR studies of 4-fluoroPhe-labeled bradykinin (18). A DANTE pulse sequence (19) was used to invert selectively either the cis or trans 19F resonances. A typical series of delays was 10 µs, 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, 1.0 s, 1.1 s, 1.2 s, and 15 s. Data were analyzed using the formalism of Perrin and Engler (20). In addition to the high temperature measurements, magnetization transfer studies were also performed at 25 °C in the presence of 10 µM recombinant human cyclophilin (Sigma). Other conditions for these studies were: 200 mM NaCl, 20 mM NaPO4, pH 7.0, 5 mM 2-mercaptoethanol, and 25% D2O. At lower temperatures, the isomerization rates were measured based on dilution of the fluorinated peptide from a concentrated solution in 0.4 M LiCl/trifluoroethanol, which has previously been reported to augment the cis/trans ratios of other proline-containing peptides (21), into the final buffer containing 1 M NaCl, 50 mM acetate-d3, pH = 5.0, and 100% D2O. A series of time-dependent spectra was obtained to determine the isomerization rate constants. The approach is discussed in greater detail elsewhere.2
Fig. 1 presents fluorescence data from studies
performed on the fluorescent peptide substrate
(Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)-Arg) (Molecular Probes), the sequence of which is derived from the Pr55gag p17/p24 cleavage site (22). Also shown are the product,
cis, and trans normalized concentrations
simulated using the kinetic model given above. As predicted, the
time-dependent fluorescence intensity exhibits clear
evidence of two distinct phases. Furthermore, other data acquired over
a range of protease concentrations (1.9-3.2 µM) also
exhibit a slow kinetic phase despite the high protease concentration.
The slow rate constant for the assay conditions shown in Fig. 1 is
determined to be 0.022 s1 by a least squares fit of this
data to a bi-exponential function. Similar values (0.022-0.023
s
1) were obtained at several protease concentrations and
at two different concentrations of NaCl (1.0 and 0.4 M).
Whereas the activity of the protease increased approximately 3-fold at
the higher salt conditions, the observed value of the slow rate
constant remained essentially constant. Thus, the slow rate constant
observed in kinetic studies such as that shown in Fig. 1 does not
correspond to a slower hydrolysis of the cis conformer, but
rather to an enzyme-independent cis
trans
isomerization.
Cis trans isomerization rates at various
temperatures were determined independently by NMR, using an analogous,
fluorine-labeled test peptide also derived from the p17/p24 cleavage
site, in which fluorine is substituted for the tyrosyl hydroxyl group:
Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln (FPhe = 4-fluoro-L-phenylalanine). The 19F NMR spectrum
of this peptide exhibits two well separated resonances (
= 0.6 ppm) at an intensity ratio of cis/trans = 0.3, which can be shown to correspond to a single, interconverting species by
magnetization transfer NMR methods. Values for the cis
trans rate constant were obtained at high temperatures
(50-80 °C) using magnetization transfer techniques analogous to
those described previously in 19F NMR studies of
4-fluoroPhe-labeled bradykinin (18). Alternatively, rate constants at
lower temperatures (0-10 °C) were obtained by initially dissolving
the peptide in a solvent (0.4 M LiCl/trifluroethanol), which has been found to augment the cis/trans
ratios of other peptides (21), followed by dilution into a 1 M NaCl buffer. The rates determined from these studies are
given in Fig. 2 as an Arrhenius plot, which shows the
expected dependence on 1000/T with
G = 17.4 kcal/mol. The cis
trans isomerization
rate constant (kc) determined using the
fluorescent peptide is shown in Fig. 2 (
), where it clearly is in
excellent agreement with the anticipated rate constant at 22 °C
determined by linear regression of the NMR isomerization data
(dashed line).
The development of protease inhibitors is based on HIV protease assays, which typically, although not always, involve cleavage of Xaa-Pro peptide bonds (22-30). In these studies and the many reported kinetic studies using this approach, it is in general not clear how the cis-trans isomerization behavior has been dealt with. The results of such assays generally depend on the enzyme and substrate concentrations and on the physical parameters utilized in the studies. Simulations performed using the trans-selective hydrolysis model given above indicate that at protease concentrations 10 times less than those used to model the data of Fig. 1, product production curves are obtained that are qualitatively close to those predicted by simple, non-selective Michaelis-Menten kinetic schemes. However, for the non-selective model, an effective catalytic rate constant must be invoked such that kcat = ftransktH, where ftrans is the fractional concentration of the peptide with trans Xaa-Pro conformation. In this limit, the formation of product peptides is also fairly well approximated by a single exponential time course according to the following equation,
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(Eq. 1) |
As discussed below, the association of cyclophilin, an enzyme with
peptidyl-proline cis-trans isomerase activity, with the Gag
substrate of HIV protease suggests that it may function as an auxiliary
enzyme to facilitate conversion of the non-cleavable, cis
peptide conformation into active trans substrate for the
protease. The cis-trans isomerization kinetics of
the fluorinated peptide in the presence of recombinant human
cyclophilin were determined at T = 25 °C using the
magnetization transfer NMR method. Typical NMR spectra observed in the
absence (panels 1) and presence (panels 2) of
cyclophilin are presented in Fig. 3. For clarity,
cis (A) and trans (B) peaks
are scaled to equal heights at equilibrium. Although isomerization of
the peptide in solution at 25 °C is normally too slow to measure
using this technique, the interconversion is readily observed in the
presence of 10 µM cyclophilin. In Fig. 3A
(panel 2), the increased isomerization rate is manifest as a
decreased net magnetization (peak height) of the cis peak as trans conformers with inverted spins rapidly convert to
cis conformers and, conversely, cis conformers
with non-inverted spins rapidly convert to the trans form.
In Fig. 3B (panel 2) the initially inverted
magnetization of the trans peak returns to equilibrium noticeably faster than in Fig. 3B (panel 1),
where the interconversion rate is slow relative to the spin-lattice
relaxation rate. Analysis of these spectra indicate that the
cyclophilin-enhanced cis trans rate, seen in
Fig. 2 (
) is significantly faster than that predicted by regressing
the peptide isomerization rates measured in the absence of
cyclophilin.
Thus, we have found that cleavage by HIV-1 protease of a peptide
related to the Pr55gag p17/p24 junction is selective for the
trans conformation of the Tyr-Pro bond, and second, that a
tyrosyl OH F peptide analog is a substrate for recombinant human
cyclophilin. Taken together, these observations suggest a possible role
of the Gag-associated cyclophilin as an auxiliary enzyme for the
protease. In vitro studies of the homodimeric enzyme show it
to be fairly unstable at pH 7 (31), and, in particular, the active
dimeric enzyme is stabilized in the presence of either inhibitors (32)
or substrates (33). Once dissociated, the monomeric unit unfolds and
becomes an excellent substrate for the active dimeric protease (34). To
the extent that peptide cleavage and protease inactivation due to dimer
dissociation are competing processes in vivo, cyclophilin could act to convert the Pr55gag p17/p24 cleavage site, as well
as the other Phe-Pro sites, from protease-inactive cis
conformations to protease-active trans conformations, thereby optimizing cleavage in the presence of an unstable protease. The association of cyclophilin with the Gag protein would appear to be
consistent with this function. Beyond the question of protease stability, the presence of cis Xaa-Pro conformations could
also alter the order of cleavage of the different sites by the
protease. The order of cleavage of the Gag-Pol scissile bonds may also
be important to achieve normal viral structure and function (35), and
the presence of cyclophilin would reduce or eliminate perturbations of
the normal order of cleavage resulting from the presence of a mixture
of cis and trans Xaa-Pro bonds. Finally, the
presence of cis conformations of target bonds could favor
proteolytic cleavage of less susceptible alternate bonds, for example
the Leu5-Trp6 bond of the protease itself (34,
36).
Navia et al. (37) have proposed that the dimeric structure of the protease may play a role in the timing of virus maturation, so that the protease is optimally active after the nascent virus particles have budded from the cell membrane, at which point the high concentration of intra-virion protease will favor the active dimer over the inactive monomer. Based on studies of the viral life cycle, Braaten et al. (17) also suggest that cyclophilin A functions within the virion, but only after proteolysis and assembly are completed. They conclude that there is some (unknown) effect of cyclophilin that is important early in the viral life cycle and that the absence or inactivation of cyclophilin leads to no biochemically detectable assembly defects. This could indicate either that cyclophilin serves another purpose in addition to a role as an auxiliary enzyme for the protease or that some very subtle structural features resulting from the presence of incompletely or incorrectly cleaved Gag-Pol become significant at a time point prior to viral-directed DNA synthesis. Further studies will be required to sort out these possibilities.