The EMBO Journal vol. 1 1 no.3 pp. 1 141 - 1144, 1992

An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase Jan Konvalinka, Magda Horejsi1, Martin Andreansky', Petr Novek, Iva Pichova, Ivo Blaha, Milan Fabry', Juraj Sedlacek1, Stephen Foundling2 and Petr Strop Institute of Organic Chemistry and Biochemistry and 1Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Flemingovo n.2, Praha 6, Czechoslovakia, and 2National Cancer Institute, Frederick Cancer Research Development Laboratories, PO Box B, Frederick, MD 21702-1201, USA

Communicated by T. Blundell

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites Si, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the BiIV-1 PR. This dramatic alteration of MAV PR activity achieved by sitedirected mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases. Key words: HIV/MAV/proteinase activity and specificity/ site-directed mutagenesis

Introduction Proteinases of retroviruses are of crucial importance for correct processing, maturation and infectivity of viral particles (Kay and Dunn, 1990). These enzymes therefore are the target of intensive research the aim of which is the rational design of inhibitors and therapeutic drugs to block viral processing, maturation and infectivity. Most of the intense effort has been directed at three proteinases, namely the AIDS virus proteinase (HIV-1 PR) (Krausslich and Wimmer, 1988) and at the two almost identical proteinases from the avian sarcome -leukemia viruses myeblastosis associated virus (MAV) (Strop et al., 1991) and Rous sarcoma virus (RSV) (Kotler et al., 1991). Structurally these enzymes are homologous, but several features distinguish HIV-1 PR from the other retroviral proteinases. HIV-1 PR shows higher activity, which may reflect a lower level of proteinase expression in lentiviruses in comparison with the C-type oncoviruses (MAV, RSV); essentially, less proteinase has to process more polyprotein. The HIV-1 PR also differs markedly from MAV and RSV in the substrate specificity and pH profile. Through site© Oxford University Press

directed mutagenesis we have identified five amino acids that are largely responsible for differences between the two enzymes, increased activity between one to two orders of magnitude, changed specificity and pH activity profile of a proteinase of a C-type retrovirus (MAV) to that of lentivirus (HIV-1).

Results The prediction of the amino acid residues forming the substrate binding pockets, S3 -S3' of wMAV PR was based on X-ray structures of the native enzymes, wMAV PR (Ohlendorf et al., 1989), HIV PR (Wlodawer et al., 1989; Lapatto et al., 1992) and RSV PR (Jaskolski et al., 1990), on the molecular model of a MAV PR-inhibitor complex and on the X-ray structure of a HIV-1 PR -inhibitor complex (Miller et al., 1989). We have chosen five amino acids of the wMAV PR and replaced them with the corresponding residues of HIV-1 proteinase sequence. In wMAV PR the following amino acids were mutated: 10OAla-Leu, 104Val-Thr, 105Arg-Pro, 106Gly-Val and 107Ser-Asn. These changes were introduced directly into the Escherichia coli expression plasmid pMG45 (Sedlacek et al., 1988), coding for a recombinant gag precursor of the wild-type MAV PR. The newly acquired specificity did not abolish the correct autoprocessing at the natural boundary between the nucleocapsid protein and the proteinase's own N-terminus. The substrate specificity of wild-type and mutant MAV proteinase was compared with HIV-1 PR on a series of chromogenic peptide substrates (Table I). The peptides 1-9 are analogues of the superior chromogenic substrate of both wMAV and HIV-1 PR (Konvalinka et al., 1990, 1991) with variations in P1, P2 and P3. This series then enables a mapping of the sequence and side-chain preference of the proteinase active clefts in subsites SI, S2 and S3. The comparison of kinetic data obtained for wMAV PR, mMAV PR and HIV PR is given schematically in Figure 1. Table I. Substrates used for subsite specificity investigation of wild-type MAV, mutated MAV and HIV-1 proteinases

Peptide no. Sequence subsite I 2 3 4 5 6 7 8 9

P6 AlaAlaAlaAlaAlaAlaAlaAlaAla-

-P5 -P4 -P3 -P2 -PlP1' ThrThrThrThrThrThrThrThrThr-

HisHisHisHisHisHisHis-

Gln- Val- Tyr* Gin- Val- Phe* Gin- Ile- Tyr* Gln- Leu- Tyr* Gln- Ala- Tyr*

Glu- ValAsp- ValHis- Tyr- ValHis- Arg- Val-

Tyr* Tyr* Tyr* Tyr*

NphNphNphNph-

NphNphNphNphNph-

-P2' -P3' -P4' -P5' ValValValValValValValValVal-

ArgArgArgArg-

LysLysLysLys-

ArgArgArgArgArg-

LysLysLysLysLys-

Ala Ala Ala Ala Ala Ala Ala Ala Ala

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k CAT/Km

Lt

30-H

20-

ro t111 ;ll m

I1

10-

Tyr peptide No. 1

-Al

II

Phe 2 P1

Val 1

I

lie 3

Leu 4

Ala 5

P2

Glu Asp Tyr Arc 6 7 8 9 P3

Fig. 1. Schematic representation of kcat over Km values for substrate series tested. For conditions

At pH 6.0 the mutated enzyme exhibits more than one order of magnitude higher activity than the wild-type, for all peptide substrates tested. Catalytic efficiency of mMAV PR as expressed by kcat over Km value is much closer to that of HIV-1 PR than to the wild-type enzyme, regardless of the amino acid residue occurring in P1, P2 or P3 of the substrate series. Detailed inspection of Km and kcat values obtained for substrate series with all three proteinases (Table II) shows that the higher catalytic efficiency of both HIV-1 PR and mMAV PR in comparison with the wild-type wMAV PR is in most cases due to beneficial changes in both parameters, i.e. increase in kcat and decrease in Km (peptides 1, 2, 3, 4, 5, 7 and 9 in Table II). The same pattern is not precisely followed in the case of peptides 6 and 8. Introduction of Tyr into P3 (peptide 8) increases kat for MAV PR 2-fold, but decreases it for both mutated MAV and HIV-1 proteinases. When potentially negatively charged Glu is introduced into the P3 position (peptide 6), the binding of wMAV PR improves dramatically compared with the 'parent' peptide 1. Some increase in binding is observed for mMAV PR too, albeit less pronounced, whilst HIV PR binds both peptides almost identically. The improvement of binding caused by a negative charge in P3 may be explained by a possible ion pair being introduced at S3. This ionic interaction is made possible by the existence of two arginines (ArglO and ArglO5) in the wMAV enzyme. The change of lO5Arg-Pro in the mMAV is not sufficient to abolish the effect entirely. All kinetic experiments previously described were carried out at pH 6.0, which falls within apparent pH optimum for 1142

see

Materials and methods.

Table II. Subsite specificity mapping of wild-type and mutated MAV proteinase compared with HIV-1 proteinase

No.of

peptide 1 2 3 4 5 6 7 8 9

Position/ residue

kcat (s l) w

m

H

P1 Tyr

5 3 2 0.2 0.5 5 1 11 3

9 7 4 1.5 2.5 5 11 7 4

7 7 5 3 2 8 3 5 5

Phe P2 Ile Leu Ala P3 Glu

Asp Tyr Arg

Km (jtM) w

m

H

82 30 42 120 9 6 25 26 24

7 13 7 25 7 3 7 2 4

4 4 4 22 16 3 2 1 2

Abbreviations used: w, wild-type MAV PR; m, mutated MAV PR; H, HIV-l PR.

all three enzymes. The pH dependence of their kinetic parameters is shown in Figure 2. This figure once again documents an activity increase of mutated MAV proteinase in comparison with the wild-type with peptide 1. Interestingly, at extreme pH regions the improvement in catalytic activity demonstrated by both kcat and Km is even larger. For the wild-type both Km and kcat values show a bellshaped pH dependence with optima at pH 6.5 and 5.5, respectively. The kcat decrease in the basic pH range is substantial (a 30-fold drop between pH 8.0 and 9.0). In contrast, the HIV PR exhibits only minor changes in kcat between pH 4.0 and 9.0 while the Km value increases steadily through the whole pH range above 6.0. Since the substrate and conditions used for both pH dependence deter-

Retroviral proteinases

PKm

log

kCAT

0.0

4

5

6

7

8

9

10

11 pH

Fig. 2. pH profile of kinetic constants for HIV-1, wMAV and mMAV proteinases.

P3' 8

.......k.....-....CA 300

.,CA300

AP

73 CAC11

Fig. 3. Stereo view of the model of mMAV PR binding cleft with a peptide substrate. P5 to P5' part of peptide substrate Thr-His-Gln-Val-Tyr-PheVal-Arg-Lys-Ala and the regions 100-110, 35-41, 300-310 and 235-241 of each monomer of mMAV PR are drawn in thick unbroken lines. The regions 100-110 and 300-310 of wMAV PR are superimposed in shaded lines.

minations were identical, this striking difference in pH profile of two so closely related enzymes must reflect important structural differences in their binding clefts. The residues

responsible for the different behaviour of the MAV proteinase may be His75 and ArgIO5, whose counterparts in HIV-1 PR are Gly48 and Pro8 1. These are situated on

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J.Konvalinka et al.

the highly mobile flap of the proteinase and d'beta strain of the structurally conserved Psi motif. Indeed, replacing of Arg 105 of MAV PR by its counterpart in the HIV-1 PR (lO5Arg- Pro) results in much flatter pH dependence of both Km and kcat for the mMAV PR. This correlates well with the HIV-1 PR dependence profiles. In an attempt to explain the substrate specificity change of the mMAV PR, we modelled the mutations into the X-ray structure of the wMAV PR, which included an energyminimized substrate and flaps for the enzyme. All mutations fall into a very flexible extended ,3-loop of the proteinase that forms part of the S1-S3, and by symmetry, S1 '-S3' specificity pockets. The model in Figure 3 suggests several changes that may be responsible for the dramatic improvement in binding of substrates in the mMAV PR. The replacement of the AlalOO for Leu diminishes the available binding area in the S2 and S2' pockets. The model demonstrates that the change of 104Val- Thr could facilitate the formation of a possible hydrogen bond between the oxygen atom of ThrlO4 and the Tyr hydroxyl at P1 of the substrates. This modelled interaction could result in tighter binding of such a substrate in comparison with a substrate harbouring Phe in P1. Moreover, the change at 106Gly-Val, reduces the available volume in SI and SI' binding pockets and contributes to the overall apolar character of the pocket. Tyr accommodated into P1 may thus bind much tighter as a result of changes in pocket volume and apolar physico-chemical property, as observed in improved Km value of peptide 1 in comparison with 2 for mMAV PR (Table II). The mutations introduced, however, are of a complex nature. Molecular modelling goes only part way to answering the questions posed by detailed kinetic experiments of this type. Only through a careful analysis of the X-ray crystal structure of the mutant enzyme and possibly of inhibitor complexes with wMAV and mMAV enzyme, could the relationship between enzyme structure and substrate recognition be defined.

Materials and methods Homogeneous wMAV PR, HIV-1 PR and mMAV PR preparations were obtained as described previously (Sedlacek et al., 1988). Construction of plasmid pMG45Ml which codes for mMAV PR was as follows: synthetic 81 bp oligonucleotide duplex carrying the indicated mutations was used to replace the wild-type sequence between the BamHI and PstI sites in the plasmid pMG45 (SedinWek et al., 1988). The peptides were synthesized by the solid phase method using Bocchemistry. Nph stands forp-nitrophenylalanine, an asterisk marks proteinase cleavage sites. The subsites are specified according to Schechter and Berger (1967), i.e. sites adjacent to the scissile bond are depicted P6-P5-P4-P3-P2-P I *P I'-P2'-P3'-P4'-P5'. The active site concentration of HIV-1 PR and wMAV PR was determined by titration with their tight-binding inhibitors (Strop et al., 1991; Roberts et al., 1990), concentration of mMAV PR was calculated from the concentration of the protein in enzyme solution. Operational proteinase concentration was in the region of 4-10 nM. Peptide substrates hydrolysed were monitored spectrophotometrically at 305 nm and 38°C as described previously (Konvalinka et al., 1991) in 0.1 M sodium acetate (pH 4.0-5.0), phosphate (pH 5.0-7.0) and Tris (pH 7.0-9.0) containing 4 mM EDTA and 2 M NaCl. Reactions were initialized by addition of a proteinase and monitored spectrophotometrically (pH 4.0-7.0) or on HPLC (pH 8.0-9.0). Peptide 1 (Table I) was used as a substrate for active-site titration.

Acknowledgements Support of this project by grant No. 45501 of the Czechoslovak Academy of Sciences is gratefully acknowledged.

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