c-tail valine is a key residue for stabilization of complex between

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 34, Issue of August 26, pp. 21467-21472, 1994

Printed in U.S.A.

C-tail Valine Is a Key Residue for Stabilization of Complex between Potato Inhibitor and Carboxypeptidase A*

(Received for publication, March 25, 1994, and in revised form, May 31, 1994)

Miguel A. MolinaS, Cristina MarinoS, Baldomero Oliva, Francesc X. Aviles, and Enrique Querolll From the Znstitut de Biologia Fonamental and Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Although the carboxypeptidase A-potato carboxypeptidase inhibitor (CPA-PCI) complex is a well known ex- ample of protein-protein interaction, little was known about the basis of its thermodynamic stability. Site-directed mutagenesis has been used to identify key residues in the PC1 tail and estimate the contribution of their chemical groups to the binding to CPA. Two deletion mutants were created, one lacking the C-terminal residue of the tail (Glf’) and another one lacking the two C-terminal residues (Val”, Glf@). The last mutant had an inhibition constant for CPA 104-fold higher than that of wild-type PCI, indicating that Vals8 is a key residue. The interactions of Vals with CPA residues contribute 5.4-5.7 kcal mol“ to the overall stability of the CPA- PC1 complex (11.9-12.1 kcal mol“). A series of PC1 point mutants at valine 38 were created, and their inhibition constant for CPA was measured. Two of these mutants with smaller side chains, V38G and V38A, allowed us to estimate that the contribution of the three side chain aliphatic groups of valine 38 to the overall stability of the complex is 3.4-4 kcal mol”. Another two mutants with larger side chains, V38L and V381, were constructed, the first being a significantly worse inhibitor than the wild type. These results suggest that only aliphatic groups in positions p and y of residue 38 in PC1 (but not those in 6) can establish van der Waals interactions with atoms of the active center of CPA and partici- pate in binding. The energetic contribution of each methywmethylene group in those positions can be estimated as 1-1.5 kcal mol”. Our hypothesis is supported by computer simulation analysis.

Some protease inhibitors are among the smallest globular proteins known. They have biotechnological and pharmaceuti- cal applications because of their involvement, together with proteases, in important processes such as peptide processing, defense mechanisms, fertilization, carcinogenesis, trauma and inflammation, virus replication, and others (Ribbons and Brew, 1976; Ryan, 1989; Billings et al., 1989; Fritz et al., 1990; Hoc- man, 1992; Aviles, 1993). The complexes of proteases with their

* This work has been supported by grants BI091-0477, BI092-0458, and IN90-0009 from the CICYT (Ministerio de Educacidn y Ciencia, Spain). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ PFPI fellowship recipient of the Ministerio de Educacidn y Ciencia (Spain). Present address: Unitat de Bioquimica i Biologia Molecular, Departament de Biologia, Facultat de Ciencies Experimentals i de la Salut, Universitat de Girona, 17001 Girona, Spain.

Q Fellowship recipient of the Programa de Cooperacih Cientifica con Iberoamerica.

ll To whom correspondence should be addressed. Tel.: 34-3-581-1233; Fax: 34-3-581-2011.

inhibitors are among the most studied cases of protein-protein interactions (Janin and Chothia, 1990), but knowledge about the molecular basis of the thermodynamic stability of these complexes is still scarce. A better understanding of the forces responsible for the protease-inhibitor association is desirable not only for theoretical but also for practical purposes, because it should facilitate the design of peptidic drugs specifically directed against particular proteins.

In this paper we present site-directed mutagenesis studies on the potato carboxypeptidase inhibitor (PCI).‘ PC1 is a small, 39-residue protein (Hass and Ryan, 1981). Its structure is known in aqueous solution (Clore et al., 1987) and in crystal complex with carboxypeptidase A (Rees and Lipscomb, 1982). The 27-residue globular core of PC1 is stabilized by three disulfide bridges and lacks regular secondary structures except for a short 5-residue helix, positions 14-18. Nevertheless, PC1 shows a defined three-dimensional structure, being a good ex- ample of a minimal globular protein. Residues 35-39 form a C-terminal tail that protrudes from the globular core. The exact biological role of PC1 is unclear, although it is probably involved in plant defense against fungal attack and phytophagous plagues (Ryan, 1989). In addition, it has been recently reported that PC1 is representative of a small cysteine-rich module, a structural fold shared by several different protein families (Holm and Sander, 1993).

PC1 can establish complexes with several carboxypeptidases inhibiting them in a strong competitive way with a K, in the nanomolar range. The best characterized complex is that with bovine carboxypeptidaseA(CPA) (Rees and Lipscomb, 1982). In this complex, the C-terminal amino acid tail of PC1 docks into the active site of the enzyme, leading to a stopper-like inhibition mechanism. In the first stages of the binding, the C-terminal residue of PC1 (GlY3’) is cleaved off by CPA, and the carboxylate group of the previous residue (VaP8) makes a coordinate bond with the active site Zn”. According to the crystal structure, all residues in the PC1 C-terminal tail make contact with CPA residues in the protease-inhibitor complex except for Gly35. The functional importance of some contact sites in the binding of PC1 to CPA has been experimentally determined by chemical modification and enzymatic studies (Hass et al., 1976; Haas and Ryan, 1980). So far, there have been no systematic studies on the basis of the thermodynamic stability of the complex and the contributions to it of specific residues and chemical groups of PCI.

In two previous studies (Molina et al . , 1992; Marino et a l . , 1994), a synthetic gene encoding the isoform IIa of PC1 was constructed and expressed in Escherichia coli using the secre- tion vector PIN-111-ompA-3, fused in frame to the OmpA signal

CPA, carboxypeptidase A , HPLC, high performance liquid chromatog- The abbreviations used are: PCI, potato carboxypeptidase inhibitor;

raphy; Kj, inhibition constant; wt, wild-type; dATPaS, deoxyadenosine 5’-a-thiotriphosphate.

21467

21468 Key Role of Val3’ in Stability of CPA-PCI Complex

TABLE I Inhibition constants (KJ of wild-type and mutant recombinant PCIs and Gibbs free energy of dissociation (AGdo) of the PCI-CPA complexes

Procedures.” Inhibition constants were calculated according to Henderson (1972) in the case of PC1 wild type and mutants delG39, V38L, V381, and The wild-type and mutant forms of PC1 were obtained as recombinant proteins in E. coli and purified as described under “Experimental

V38F and according to the Lineweaver-Burk method in the case of PCIs V38A, V38G, and delV38G39. Several independent determinations of the inhibition constant were made for each form of PCI. The dissociation free energy of the PCI-CPAcomplexes was calculated according to the formula AG: = -RT In K,

Inhibition constant, K, AG: of PCI-CPA complexes Complex

Interval Mean Interval Mean

M” kcal mol” Wild-type PCI-CPA PCIdelG39-CPA

0.9-2.1 x 10-9 1.5 x 10-9 11.6-12.1 11.8 3.1-4.3 x 10-9 3.7 x 10-9 11.3-11.5 11.4

65-95 x 10-9 76 x 10-9 9.4-9.6 PC1 V38A-CPA PC1 V38G-CPA 0.74-0.9 x

9.5 0.82 x 8.1-8.2 8.1

5.9 PCIdelV38G39-CPA 26-55 x loe6 11.4-14.7 x IO‘$ PC1 V38L-CPA

PC1 V38I-CPA

41 x 10“j 5.7-6.1 13.0 x 10-9 10.5-10.7 10.6

1.7-2.5 x 10-9 2.1 x 10-9 11.5-11.7 11.6 PC1 V38F-CPA 0.9-2.1 x 1.5 X 10-9 11.&12.1 11.9

peptide-encoding sequence. The recombinant PC1 was found almost exclusively in the culture medium, not in the periplas- mic space, as would be expected from OmpA signal peptide fusions. In the present work we report the analysis, by site- directed mutagenesis, of the role of the PC1 residue Val38 and its side chain chemical groups in the stabilization of the CPA- PC1 complex. Our results indicate that both the side chain and the main chain chemical groups of V a P make a major contribution to the energetics of the CPA-PC1 interaction.

EXPERIMENTAL PROCEDURES Chemicals and Enzymes-Enzymes were purchased from Boehringer

Mannheim and Pharmacia Biotech Inc. [ C ~ - ~ ~ S ] ~ A T P C ~ S was obtained from Amersham Corp. M13 sequencing kits were from Pharmacia (T7

Amersham kit. sequencing kit). Site-directed mutagenesis was performed using the

Cloning, Site-directed Mutagenesis, and Gene Expression-The con- struction of a synthetic gene for PCI, its expression in E. coli, and a procedure to purify recombinant PC1 secreted into the culture medium have been previously reported (Molina et al., 1992; Marino et al., 1994). The synthetic gene for PC1 was cloned in the pINIII-ompA-3 vector (Ghrayeb et al., 19841, fused in frame to the ompA signal sequence, generating the vector pIMAM3. Mutagenesis and sequencing were performed after cloning the XbaI-EcoRI fragment of pIMAM3, comprising the PC1 gene and the OmpA signal peptide, in vectors M13mp18 and M13mp19 (Yanisch-Perron et al., 1985). The strain TG1 (Gibson, 1984) was used as a host for propagation of MI3 derivatives. Site-directed mutagenesis of the PC1 gene was done according to the method of Nakayame and Eckstein (1986). The designated deletions and point mutations were delG39, delV38G39, V38G, V38A, V38L, V381, and V38F. Mutant PC1 genes were recloned in the PIN-111-ompA-3 vector. Sequence analyses were performed by the dideoxy method (Sanger et al., 1977).

The E. coli strain MC1061 (Casadaban and Cohen, 1987) carrying the pIMAM3 plasmids was used to produce wild-type and mutant recombinant PCIs, as previously reported (Molina et al., 1992). Wild-type and mutant inhibitors were found in the culture medium. Cultures were harvested after 24 h by centrifugation (10,000 x g, 20 mid, and the supernatant (extracellular medium) was kept for repurification.

Zmmunodetection-Polyclonal purified antibodies against PC1 were used to detect it in enzyme-linked immunosorbent assays during pro- duction and purification of wild-type and mutant forms of the inhibitor. They were obtained as previously reported (Molina et al., 1992).

Purification and Characterization of Wild-type and Mutant PCIs- Wild-type and mutant forms of PC1 were purified from extracellular medium of E. coli (pIMAM3) cultures by ion exchange on DEAE fast protein liquid chromatography and reverse phase on C18 HPLC as previously reported (Molina et al., 1992). PC1 was detected by inhibitory measurement assays, according to Hass and Ryan (1981), and by enzyme-linked immunosorbent assay. The concentration of the purified solutions of recombinant PCIs was determined from the A,,, of the final solution (PC1 extinction coefficient, E,,,, = 3.0) and also by Bradford assay using wild-type PC1 as a standard. Both methods gave the same results. Molecular masses were confirmed by mass spectrometry in a Kratos Kompact MALDI 3 V2.0 spectrometer.

The K, values of the PC1 wild type and mutants delG39, V38L, V381, and V38F for the inhibition of bovine CPA were determined according to the method of Henderson (1972). K, values of mutants PCIdelV38G39, V38G, and V38A were calculated according to the Lineweaver-Burk method. Benzoyl-glycyl-L-phenylalanine was used as a substrate at different concentrations. For each, several measurements of varying PC1 concentration were made. Enzyme (bovine carboxypeptidase A) was 42.5 nM in all cases. Substrate hydrolysis was followed by A,,, measurements made every 20 s for 2 min. Velocities were expressed as the slope of the linear increase in absorbance and used to calculate K,. Several independent K, determinations were performed for wild-type and mutant PCIs. The average K, for each form of PC1 and Gibbs free energy (AG,’) for dissociation of the PCI-CPA complexes were calculated.

Computer Simulation and Graphics-Molecular graphics and simu- lations were performed on a Crimson Elan, from Silicon Graphics. The structure of wild-type PC1 was taken from the x-ray structure of the PCI-IIa isoform in the complex with CPA (Rees and Lipscomb, 1982), adding the C-terminal residue Gly. The structures of the PC1 mutants and the corresponding complexes with CPA were modeled and visual- ized from the wild-type PC1 structure by means of the TURBO FRODO program (Roussel and Cambillau, 1991). These structures were optimized by 5000 steps of steepest descent using the GROMOS package under the noninertial solvent (NIS) force field (van Gunsteren and Berendsen, 1987). The position of the main chain atoms was constrained, assuming that conformational changes in mutants are small and do not significantly perturb the structure of PC1 (see “Results”). Calculation of the volumes of the optimized structures were made using the GEPOL program (Pascual-Ahuir et al., 1987).

RESULTS

To test the role of Val38 in the stability of the PCI-CPA complex, a series of site-directed mutants was created: delG39, delV38G39, V38G, V38A, V38L, V381, and V38F. All of these mutants were expressed in E. coli as extracellular soluble proteins. They showed chromatographic behavior in ion-exchange and reverse-phase HPLC identical to that of wild type. This fact suggests that all of them have the same overall conformation and that amino acid replacements do not alter the disulfide pairing and the folding of PCI.

All of the mutants showed competitive inhibitory activity toward CPA although with very different K,. Table I shows the K, values for CPAof all of the mutant forms of PC1 as well as the AGdo of the complexes calculated according to the equation AG,” = -RT In K,. This calculation is feasible because in the case of competitive inhibitors such as PC1 the inhibition constant is a true dissociation constant for the enzyme-inhibitor complex (Todhunter, 1979; Palmer, 1985). The K, value for the wild-type recombinant PC1 was 0.9-2.1 x lo-’ M, corresponding to a AG,” for wild-type PCI-CPA complex of 11.6-12.1 kcal mol-’. This value differs from that derived by Rees and Lipscomb (1982),11 kcal mol-’, because they used for their calculation the first K, value reported for PCI, 5 x lo-’ M (Ryan et al., 1974). Later, the K, was reevaluated by the same authors to 1.5-2.7 x lo-’ M

Key Role of VaP8 in Stability of CPA-PCI Complex 2 1469

(Hass and Ryan, 1981) corresponding to a AG: of 11.7-12.0 kcal mol-l.

The K, of the first mutant of the series, delG39, was 3.1-4.3 x lo-' M, only slightly higher than that of wild-type PCI, 0.9-2.1 x lo-' M. This fact indicates that the last residue of the C-tail of PCI, Gly39, has no important role in the stabilization of its complex with CPA. In fact, a highly homologous inhibitor from tomato lacks the C-terminal glycine (Hass and Hermodson, 1981). In contrast, the mutant PC1 with deletion of the last two residues of the C-tail, VaP8 and Glf9 (PCIdelV38G39), showed a dramatic decrease of inhibitory activity toward CPA (its K, increased about lo4 times with respect to that of PCIdelG39). The stability of the PCIdelV38G39-CPA complex, expressed as AG:, decreased to 5.7-6.1 kcal mol" (see Table I). The overall contribution of VaP8 to the stability of the PCI-CPAcomplex can be estimated by comparing the AGdo obtained for the PCIdelV38G39-CPA complex with that of the PCIdelG39-CPA complex, because the differences in the respective K, values and stabilities of the complexes are attributable to the absence of the residue Val38. We can therefore estimate that V a P contributes 5.2-5.8 kcal"mo1" to the overall stability of the natural complex. This value represents about half of the total stability of the complex, 11.6-12.1 kcal mol-l, confirming that VaP8 is a key residue for the inhibitory activity of PCI.

The low inhibitory activity shown by PCIdelV38G39 prompted us to obtain the point mutants V38A and V38G to estimate the contribution of the interactions established by the different side chain groups of PC1 VaP8 with residues at the CPA active site. The large increase in the inhibition constants observed for both mutants (Table I) with respect to the wild type, from 0.9-2.1 nM to 65-95 nM and 740-920 nM, respectively, clearly indicates that the side chain aliphatic groups of Val38 play an important role in the binding to CPA. The mutant V38G cannot establish with CPA those interactions involving the three aliphatic side chain carbons of VaP8. Therefore, the difference in AG: between the complexes of CPA with PC1 V38G and with wild-type PCI, 3.4-4 kcal mol-', is an estimation of the hydrophobic contribution of these three aliphatic carbons to the overall stability of the complex (Fig. la). As previously discussed, the total energetic contribution of Val38 is 5.2-5.8 kcal mol-'. The difference between this contribution and the hydrophobic contribution of the side chain is 1.2-2.4 kcal mol-', which can be attributed to the other interactions that Val38 establishes with CPA (two hydrogen bonds and a coordinate bond with the Zn2+ atom of the enzyme (Rees and Lipscomb, 1982). Estimations of the free energy of hydrogen bond formation, based on studies with mutants, gave values of 0.5-2.0 kcal mol-' per hydrogen bond (Serrano et al., 1992). Our results are consistent with these estimations.

The second mutant, PC1 V38A, showed a free energy of dissociation with CPA of 9.P9.6 kcal mol". The only difference between mutants V38G and V38A is the side chain of residue 38 (a hydrogen atom and a methyl group, respectively) (Fig. la). The difference in Ki and stability of the PCI-CPA complex between both mutants is, therefore, attributable to the hydrophobic contribution of the side chain methyl group in position 38 of PC1 V38A. This contribution can be estimated as 1.2-1.5 kcal mol-'. Because this methyl group is equivalent to the methylene in position P of VaP8 in the wild-type PCI, it can be assumed that the contribution of the latter group to the overall stability of the wild-type PCI-CPA complex is also 1.2-1.5 kcal mol-'.

The difference in AGd0 between the complexes of wild-type PCI-CPAand PC1 V38A-CPAis 2-2.7 kcal mol-', attributable to the two y-methyl groups of Val3*, absent in the mutant PC1 V38A (Fig. 1, a and b) . Because both groups are sterically

a PC1 W-CPA 11.6-12.1 Kcallmol

PC1 V U - C P A 10.5-10.7 Kcallmol

0.9-1.6 Kcallmol

2-2.7 Kcallmol

I Whole side-chain contribution 3.4-4 Kcallmol

PC1 VRRA-CPA 1

1 L3-CH2 I 9.4-9.6 Kcallmol

1 i.2-1.5 Kcallmol I PC1 V38G-CPA 1 f

8.1-8.2 Kcallmol

b G ~ Y Ala Val Leu Ile Phe

contribution limit

plexes formed by CPA and the recombinant PC1 wild-type, V38L, V38A, FIG. 1. a, differences in dissociation free energies (AG:) of the com-

and V38G mutants. The energetic contributions, estimated from these differences, of each aliphatic group of the VaP side chain in the overall stability of the PCI-CPA complex are indicated. b, schematic represen- tation of the side chain groups of the residue in position 38 in PC1 V38G, V38A, wild type, V38L, V381, and V38F. According to our hypothesis, only aliphatic groups in positions and y establish hydrophobic interactions with atoms of the active site of CPA in the protease-inhibitor

V381, do not establish such interactions and therefore do not contribute complex. Methyl groups in position 6, present in mutants V38L and

to the stability of the PCI-CPA complex.

equivalent, they are likely to contribute equally, 1-1.4 kcal mol", to the overall stability of the PCI-CPA complex. This value is similar to the contribution of the P-methylene of Val38 deduced above. These results suggest that the hydrophobic contribution of each methyVmethylene group of residue 38 to the AGdo of the PCI-CPA complex is the same, about 1-1.5 kcal mol".

The mutants so far analyzed indicated that the side chain hydrophobic contribution of the residue in position 38 of PC1 is very important for the stability of the protease-inhibitor complex. In order to find out if it was possible to obtain a better inhibitor by increasing this hydrophobicity, we constructed three more mutants: V38L, V381, and V38F. Surprisingly, the mutant V38L was less active than wild-type PC1 (the free energy of the PC1 V38L-CPA complex is 10.5-10.7 kcal mol"). In contrast, the mutant V38I presented a K, indistinguishable from that of wild-type PCI. This fact was unexpected because both leucine and isoleucine side chains have four methyl/ methylene groups, varying only in their positions. In order to explain these results, we formulated the hypothesis that the stability of the PCI-CPA complex depends not on the overall side chain hydrophobicity of residue 38 of PC1 but only on the additive hydrophobic contributions of the methyYmethylene groups in positions P and y (Fig. l b ) . According to this hypothesis, the aliphatic groups in position 6 of residue 38 of PCI, present in mutants V38L and V381, would not make any contribution to the stability of the PCI-CPA complex.

Leucine has a methylene in the P position and another in the y position, whereas valine has one methylene in P and two methyls in y. If the above hypothesis is right, the CPA complex with V38L should have a AGdo 1-1.5 kcal mol" lower than that of the complex with wild-type PCI, because it loses one of the methylene groups in y that contributed 1-1.5 kcal mol" to the binding. The difference found experimentally between the AG: of the two complexes was 0.9-1.6 kcal mol" (Table I), in perfect

21470 Key Role of VaP8 in Stability of CPA-PCI Complex

agreement with the value predicted according to our hypothesis. This hypothesis also allows us to explain the K, of the mutant V38I. Valine and isoleucine have the same number of methyurnethylene groups in the p and y positions of their side chains. Therefore, their complexes with CPA should have the same AGdo, and that is exactly what was experimentally found (Table I and Fig. 1).

A computer simulation was performed to gain insight into the thermodynamic basis of the binding affinities of the different PC1 variants in residue 38. The mutants in the complexes were modeled starting from the wild-type PCI-CPA complex x-ray structure. The positions of the main chain atoms of PC1 were constrained, the mutant side chains were built over them, and an optimization of the structure by energy minimization was performed to avoid nonallowed contacts of the mutated side chains. This approach assumes that the conformational changes in mutant PCIs are small and do not significantly perturb positions of the main chain atoms of PC1 in the PCI- CPA complex. First, the inhibitor establishes many contacts with CPA (Rees and Lipscomb, 1982), and only a few of them are directly affected by mutations of Val38; but more impor- tantly, all of the mutants conserve a considerable (competitive) inhibitory power, particularly those carrying the bulkiest residues. Their differences in K, can be interpreted exclusively by changes in interactions involving side chain atoms of residue 38. In addition, the excess of volume at side chain 38 is accom- modated at the large active site cavity, as shown below. After energy minimization the surface triangulation program GE- POL (Pascual et al., 1987) was used to calculate the volumes of wild-type and mutant PCIs and of their complexes with CPA in order to calculate the differences in volume between mutants and their complexes. The excluded volume when forming the complex, Vexel, was calculated according to the expression,

v'"l = vCpA + vP,I - vCPA.pCI (Eq. 1)

where V,, is the volume of CPA alone, V,,,, is the volume of PC1 alone, and V,,,,, is the volume of the complex.

The mutations of residue 38 only affected the hydrophobicity and volume of its side chain. Therefore, we assume that mainly the entropic factors of the interactions between PC1 and CPA were perturbed. Computer graphic analysis of the energy-mini- mized complexes showed that the electrostatic interactions and hydrogen bonds between the molecules were essentially main- tained in the whole series. This is an obvious consequence of the model building employed. Subsequently, we compared the changes in volume with the variation in hydrophobicity, that is with the tendency of the side chain to become buried, using the solvation free energy values of Eisenberg and McLachlan (1986). As shown in Fig. 2a, there is a clear relationship between the volume increase of PC1 alone and the corresponding increase in the solvation free energy of the different residues placed at position 38 of PCI. In contrast, the total volumes of the PCI-CPA complexes are only affected for mutant residues Ile or Leu, that is for those containing &methyl groups. The change is even larger for the V38F mutant (not included in Fig. 2; this particular case will be discussed later).

If our approach is valid, there should be a linear relationship between the change in dissociation energy of the complexes A(AG) relative to the wild-type PCI-CPA complex and the increase in the excluded volume. We can calculate the change in dissociation energy of the complexes A(AG) relative to the wild- type PCI-CPA complex as

A(AG) = -k(V$' - VZI) = - k AV""' (Eq. 2)

where k = KTp (Richmond, 1984), V F ' is the excluded volume when forming the CPA-mutant complex, V$' the excluded vol-

a

- 8.20 Ile

l inhibi for ]

Ala P

63.60 ' I 8 ; 8.00

0.0 0.5 I .o 1.5 2.0 2.5 Solvation Free Energy (kcal mol.')

b

4'00 F 3.00 ;

7- - F 2.00 1

V38G 0

v38/

?2 ,- 1.00

-1.00 0.0 20.0 50.0 80.0 110.0

- AV'''' (nm3 X 1 03)

and in complexes with CPA (left), uersus the solvation free energies of FIG. 2. a, total volumes of wild-type PC1 and mutants, alone (right)

mutated residue 38 of PCI, according to Eisenberg and McLachlan (1986). b, increase of AG," of CPA-PC1 complexes versus the difference of excluded volumes. The slope of the linear regression (rn = 0.04) lies within the range of the predicted value derived from Equation 2 ( K T p ) (see "Results"). V,, excluded volume of the wild-type PCI-CPA complex; P C ' , excluded volume of the mutant PCI-CPA complex.

ume when forming the wild-type PCI-CPA complex, and AVexc' is the difference in the excluded volume.

A linear relationship was found between A(AG) of the complexes calculated from the experimental data and the increase in the excluded volume for the different PC1 variants (Fig. 2b). All of these results strongly support our hypothesis that any side chain aliphatic C6 in position 38 is not buried in the active site cavity of CPA, is probably in contact with the water shell of the complex, and is not making any contribution to its stability.

Analysis of the generated structures of the different mutant inhibitor-CPA complexes in computer graphics helps us to vi- sualize and understand our results. The S1 subsite position in the enzyme, which is located in a narrow passage close to residue 38 of PCI, is followed by a wide pocket. Only chemical groups in positions p and y of residue 38 would be in appropriate positions to be buried in the active center of the CPA, whereas C8 in mutants V38I and V38L faces the water shell. In the case of mutant V38L, one of the y-methyl groups of Val3' is absent. In wild-type PCI, this y-methyl interac!s with the aromatic-rings of two CPAresidues: Tyrlg8 (at 3.79 A) and Phe279 (at 3.81 A). In the case of mutant V381, in which both y-methyl groups are present, the K, was similar to that of wild-type PCI. Fig. 3 shows a stereo view of one model-built mutant of PCI, Ile38, in the S1 subsite of CPA. It can be seen that the 8-methyl group of the mutant is directed backward to the PC1 core, specifically to TrpZ8 of PCI, and has no significant contacts with CPA. Therefore, it cannot contribute to the stability of the complex.

Key Role of Val38 in Stability of CPA-PCI Complex 21471

FIG. 3. Computer graphics visualization of the model-built mutant PC1 V38I interacting with CPA in the S1 subsite. The stereo view depicts the spline Connolly surface (rolling sphere of 1.4 A) of CPA (thin white lines). Wild-type PC1 is displayed as a white ball and stick model, and the mutated Ile38 side chain is dark. Computer graphics representa- tions were produced using the TURBO FRODO program.

The mutant V38F is worth a separate discussion. I t showed a Ki indistinguishable from that of wild-type PC1 (Table I). This residue was not included in the graphs of Fig. 2 because, in addition to the entropic effects, it shows a significant enthalpic contribution ( P interactions) with nearby aromatic residues. This is why the hypothesis we used to explain the results obtained for the other mutants cannot be used in this case. Phen- ylalanine has just one aliphatic Cp plus an aromatic ring. The contribution of this ring to the stability of the PC1 V38F-CPA complex can be estimated as 2-2.7 kcal mol-', that is the difference in AG: between CPA complexes with PC1 V38A and with PC1 V38F. Again, visualization by computer graphics helps to understand the experimental results. Computer graphics analysis shows that the Phe3' phenyl group of the mutant does not seem to alter the structure and stability of the complex because it lies between the two nearby aromatic side chains of w4' of CPA and of Trp2' of PCI, the three rings being kept oriented in appropriate angles to make favorable P interactions. Our estimation of the aromatic ring contribution, 2-2.7 kcal mol-', is in line with the reported range for the energetic contribution of interactions between two aromatic rings in the stability of proteins (Hunter et al., 1991).

DISCUSSION Previous knowledge of the relative or quantitative contribu-

tion of PC1 residues to the inhibition of carboxypeptidase A was limited. It was based on chemical modification and enzymatic studies (Hass et al., 1976; Hass and Ryan, 1980) and on the analysis of contact areas in the crystal structure of the complex (Rees and Lipscomb, 1982). These studies attributed the binding and inhibitory ability of PC1 to the primary and secondary contact regions of the inhibitor without a deeper appraisal of their contributions. They also attributed a minor role to GlY9. Although this general view is still valid, from the present study we can conclude that a single residue of PCI, Val3', is a key residue because it contributes as much as the rest to the binding and inhibition. Thus, 4548% of the free energy of the binding, 5.2-5.8 kcal mol-' over 11.6-12.1 kcal mol-', is attributable to this residue. This is a relevant result considering that the other 9 residues of PC1 primary and secondary contact regions have been shown to establish defined contacts with the enzyme in its crystal structure.

Our site-directed mutagenesis studies also show that the decrease in binding energy after Val3' removal cannot be exclusively attributed to the loss of the interactions of its carboxylate

with the Zn2+ and with w4* of the enzyme (interactions visu- alized in the crystal complex) but also can be attributed to the loss of its side chain groups. Thus, 3.4-4 kcal mol" of the PCI-CPA complex dissociation energy are attributable to the hydrophobic interactions of the Val3' side chain with residues of CPA, the contribution of each of the methyllmethylene groups of this side chain being 1-1.5 kcal mol". This estimate fits well into the range of values reported elsewhere for the contribution of aliphatic groups to the stability of proteins (Kellis et al., 1989; Matsumara et al., 1989; Pace, 1992). The importance of Val3' in the binding of PC1 to CPA is probably related to the important role of the subsites locally involved (P1 in the inhibitor and S1 in the enzyme). Both subsites are essential in the recognition, binding, and catalysis of oligopeptide substrates and inhibitors by CPA (Abramowitz et al., 1967; Christianson and Lipscomb, 1989). In this respect, it is worth mentioning that the S1 subsite shows the higher degree of stereospecificity between the enzyme subsites. This is the region in which the substrate is submitted to a torsional strain that facilitates catalysis (Nakagawa and Umeyama, 1978).

The replacement of the Val3' side chain by shorter or longer apolar ones in mutant PCIs affects the inhibition constants in a trend that suggests that this side chain should have a precise size (ie. that of Val) to efficiently bind to CPA. The analysis of the series of mutants in this residue shows that the stability of the complexes with CPA depends not on the overall side chain hydrophobicity of residue 38 but on the hydrophobic contributions of the methyllmethylene groups in positions p and y. Computer simulation of the variation of the total volumes of PC1 and complexes versus solvation free energies or versus the experimental changes in stability, together with the computer graphics inspection of the interactions at the S1 subsite of CPA, corroborates our conclusions. This indicates that side chain groups in position 6 of residue 38 would not contribute to the stability of the CPA-PC1 complex because they would be oriented out of the narrow passage of the CPA S1 subsite, not buried in the active center of the enzyme.

It has been reported that it is difficult to formulate simple general correlations between dissociation energies of protein- protein complexes and interactions in contact surfaces because of the fact that the energetic contributions of certain kinds of interactions ( i e . hydrogen bonds and hydrophobic interactions) are not isotropic but are dependent on the geometry and envi- ronment surrounding the interacting groups (Janin and Cho- thia, 1990; Walls and Sternberg, 1992). Nevertheless, in par-

21472 Key Role of Val3' in Stability of CPA-PCI Complex

ticular cases such as ours it is possible to find simple correlations between interactions in contact surfaces and dissociation energies of protein-protein complexes, such as the correlation found between the number of aliphatic carbons in positions /3 and y of PC1 and the AG: of PCI-CPA complexes. Finding such correlations could represent a way of achieving an important goal in protein engineering: the rational improve- ment of the properties of proteins and protein-ligand complexes.

Acknowledgment-We are grateful to M. Lockwood for revision of the manuscript.

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c-tail valine is a key residue for stabilization of complex between

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