(Received for publication, February 9, 1996)
From the
The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave new substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally.
Rational design of HIV-1 ()protease inhibitors as
therapeutic agents for AIDS will require a thorough understanding of
the molecular mechanisms that govern the complex interactions between
the enzyme and its substrates. This necessitates identification of key
amino acids that determine substrate specificity. To identify these
critical residues, we have exploited differences in structure and
specificity between the related proteases from Rous sarcoma virus (RSV)
and HIV-1. Although these two enzymes share a 30% amino acid identity
and common overall topology(1, 2) , they possess
markedly different substrate specificities. HIV-1 protease catalyzes
the cleavage not only of its own Gag and Gag-Pol polyprotein sequences
but also those of the native RSV protease (PR)
substrates(3, 4) . In contrast, RSV PR has a more
limited substrate range, cleaving its own, but not HIV-1 PR,
polyprotein sequences. To gain insight into the basis for these
differences, we identified protease residues located within 10 Å
of a bound substrate positioned by analogy to x-ray crystal structures
of HIV-1 PR-inhibitor complexes(5) . Alignment of the two
enzyme structures revealed that many amino acid residues in the RSV
substrate binding pockets are identical to those in the structurally
equivalent positions of HIV-1 protease. However, a small number of
structurally equivalent residues differ between the two proteins (Fig. 1). We hypothesized that these amino acid differences
contribute to the difference in substrate preference between RSV and
HIV-1 proteases. To test this idea, a number of RSV protease mutants
were constructed by site-directed mutagenesis that replaced one or two
of these RSV residues with the structurally equivalent HIV-1 residues (3, 4, 5, 6) . Many of these
constructs were active and displayed partially altered specificity for
substrate selection at one or two of the eight enzyme subsites.
Consistent with this result, Sedlacek et al.(7) also
showed that some of these changes allow for partial HIV-1-like
specificity with modified peptide substrates. Recently, several of
these residues have been shown to be substituted in viral mutants that
arise during the development of drug resistance to HIV-1 protease
inhibitors in clinical trials with AIDS patients(8) . For a
review of the resistant phenotypes, see (9) . Changes in
substrate preference obtained when single amino acid substitutions were
introduced into RSV PR were sufficient to allow for some catalytic
activity on peptides that represented one or two HIV-1 polyprotein
cleavage sites(3, 4, 5, 6) .
However, it became clear that because subsites were acting somewhat
independently in substrate selection, multiple amino acid substitutions
would be required to affect a complete change in enzyme specificity. In
this report, multiple amino acid changes in enzyme subsites have been
combined into one construct and shown to impart substantial HIV-1-like
behavior upon the RSV PR. Moreover, a covalently linked dimer PR was
used to demonstrate that the symmetric subunits of the enzyme recognize
both halves of a substrate equally.
Figure 1: Schematic representation of the RSV NC-PR substrate, PAVSLAMT, from P4 to P4` in the S4-S4` subsites of PR. The relative size of each subsite is indicated approximately by the area enclosed by the curved line around each substrate side chain. Protease residues forming the subsites are shown for those that differ between the RSV and HIV-1 PRs. RSV PR residues are shown outside the parentheses, whereas the HIV-1 PR residues are shown within the parentheses. Most of the residues contribute to more than one adjacent subsite and this is indicated by the relative positions of the labels.
Figure 2:
Bacterial expression and purification of
RSV PR. RSV PR (wild-type) was expressed and purified as described
under ``Experimental Procedures.'' Soluble extract from
induced cells (lane 1) was passed over a Ni-NTA column. PR was
eluted with a 250 mM imidazole wash (lane 2). The
NH-terminal histidine tag was removed by treatment with
bovine factor Xa. After treatment, the PR was applied to the Ni-NTA
column which was washed with buffer (lane 3) and then 30
mM imidazole wash (lane 4). Factor Xa was then
removed by treatment with immobilized benzamidine (lane 5).
Protein molecular mass markers 143, 97, 50, 35, 30, and 22 kDa are
shown (lane M). The molecular mass of native RSV PR monomer is
13.5 kDa.
Figure 3: Stereo representation of the RSV PR containing nine amino acid substitutions that influence the selection of substrate and catalytic rate. Amino acids important for substrate selection and catalytic rate by RSV protease are shown by ball and stick structures in a crystal structure model of a homodimeric enzyme. These include RSV PR positions 38, 42, 44, 73, 100, 104, 105, 106, and 107. The RSV NC-PR peptide substrate is shown in the active site with thick lines. The figure was prepared with MOLSCRIPT(15) .
There are two additional indicators that the RSV(S9) PR has substantial HIV-1 PR character. First, it is inhibited effectively by the previously described nanomolar HIV-1 PR inhibitor, KNI-272(14) . At inhibitor concentrations which completely block activity of both HIV-1 PR and RSV(S9) PR, wild-type AMV PR is unaffected (Fig. 4). Second, the salt dependence of RSV(S9) PR is closer to that observed with HIV-1 PR than that with RSV PR (Fig. 5). For instance, in the presence of 1 M NaCl, HIV-1 PR has 100%, RSV(S9) PR has greater than 60%, and the AMV PR has less than 20% of their respective maximal activities with the NC-PR peptide substrate.
Figure 4:
Inhibition of AMV, RSV(S9), and HIV-1 PRs
by the HIV-1 PR inhibitor KNI-272. Ten ng of AMV (), RSV(S9)
(
), or HIV-1 PR (
) was incubated with 100 µM NC-PR peptide and various concentrations of KNI-272. Activity is
expressed as a percentage of activity in the absence of
inhibitor.
Figure 5:
Effect of salt on the activity of AMV,
RSV(S9), and HIV-1 PRs. PR activity was measured as described in the
legend to Fig. 4, except that the amount of the NaCl in the
assay was varied as indicated. , HIV-1 PR;
, RSV(S9) PR;
,AMV PR.
While RSV(S9) PR displays substantial HIV-1
PR-like substrate specificity and kinetics, complete conversion to
HIV-1 PR specificity has not been reached. This is seen by differences
in activity between HIV-1 and RSV(S9) proteases with HIV-1 substrates (k/K
values in Table 1), differences in effectiveness of the KNI-272 inhibitor (Fig. 4), and salt dependence for activity (Fig. 5). One
of several possible reasons for this is that the nine amino acids
substituted into the RSV PR influence substrate amino acid selection
primarily in six of the eight enzyme subsites, S3 to S3`. These
substitutions have limited influence on substrate selection in the S4
and S4` subsites. An additional mutation that deletes RSV PR residues
61-63, at the base of the enzyme flaps, alters preference for
amino acids interacting with the S4 and S4` subsites to resemble that
of HIV-1 PR(4) . These residues are unique to the RSV enzyme,
which has larger flaps than the HIV-1 PR. Unfortunately, when this
deletion was combined with other RSV PR mutations, it produced an
inactive enzyme. It seems likely that the removal of these residues
caused a conformational change which was not tolerated in the context
of the other mutations. Additional changes in the RSV(S9) PR will
probably have to be made in order to accommodate the S4 and S4`
deletions.
Analysis of RSV(S9) PR activities provides some insight into protease substrate recognition. One can explain the varied steady state kinetic data with different HIV-1 substrates in Table 1by the fact that the peptide substrates each have markedly different amino acid sequences, and the (S9) mutations do not affect cleavage at each site equally. Thus, strong interactions between the enzyme and the CA-NCb, CA-NCa, and NC-p6a substrates may depend not only on differences in the S3 to S3` subsites, but also on the S4 and S4` subsites that were not altered in the RSV PR(S9). In contrast, RT-IN and inhibitor KNI-272 interactions do not seem to depend on changes in the S4 and S4` subsites.
Figure 6: Schematic representation of the effects of asymmetric S2 subsite PR mutations on cleavage of NC-PR peptide substrates with leucine substitutions in the P2 and/or P2` positions. The noncovalently linked PR is indicated at the top of the figure by the presence of two separated subunits (A and B). The covalently linked PR is indicated by subunits connected with the triangle at the top (C and D). The wild-type S2 and S2` subsites are represented by the small half-circles. S2 and S2` subsites with the I42D,144V specificity altering substitutions are represented by the large half-circles. A diagrammatic representation of the NC-PR peptide substrate is depicted below with wild type residues in P2 and P2` denoted by small circles and Leu substitutions by the large circles.
Figure 7: Effects of S2 subsite substitutions in different subunits of the PR dimer on cleavage of NC-PR substrates with amino acid substitutions in the P2 and/or P2` positions. Changes in protease substrate preference caused by substitutions in the S2 enzyme subsite in either one or both subunits was determined. Enzyme activity was measured using a RSV NC-PR peptide substrate with Leu substituted in P2 (PPALS-LAMTMRR) (gray boxes), in P2` (PPAVS-LLMTMRR) (black boxes), or in both P2 and P2` (PPALS-LLAMTMRR) (white boxes). Activity is expressed as a percentage relative to the initial rate of cleavage of the wild-type NC-PR peptide substrate and was measured using the fluorescamine assay(4) . PRGGGGPR is a wild-type RSV PR that has the two subunits of the homodimer linked with four Gly residues. RSV PR (I42D,I44V) is a noncovalently linked protease homodimer with substitutions in the S2 and S2` subsites. RSV PRGGGGGPR (I42D,I44V(N)) and RSV PRGGGGGPR (I42D,I44V (C)) are covalently linked homodimers with asymmetric substitutions in either the amino or carboxyl subunits as indicated by the N and C, respectively. The activity data are summarized in Fig. 6, A-D.
The results presented here demonstrate that amino acids can be substituted at key residues in most of the enzyme subsites that alter RSV PR specificity. The present construct combined nine separate amino acid substitutions and produced an RSV PR that cleaves peptide substrates representing HIV-1 gag and pol gene cleavage sites. Furthermore, by using a covalently linked PR dimer, we have demonstrated that key amino acid residues can be changed in separate subunits to produce an asymmetrically substituted enzyme. This strategy can be used to create a PR, targeted to a new protein sequence, in which each enzyme subunit is custom designed to bind efficiently to one-half of the new substrate sequence. The resulting enzyme will prefer one substrate orientation over the reverse orientation. Finally, examination of structural differences between similar yet catalytically unique enzymes has advanced our understanding in a way that would not have been possible if each had been examined individually. This general approach can be extended to many other protein families to identify key enzyme residues that mediate specific protein-substrate and protein-protein interactions.