C-tail Valine Is a Key Residue for Stabilization of Complex between ...

Download PDF
3 downloads 0 Views 2MB Size Report
Comment
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. ..... Computer Simulation and Graphics-Molecular graphics and simu- lations were ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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 butalso for practical purposes, because it should facilitate thedesign of peptidic drugs specifically directed against particular proteins. In this paper we present site-directed mutagenesis studies on the potatocarboxypeptidase inhibitor (PCI).‘ PC1 is a small, 39-residue protein(HassandRyan, 1981). Itsstructureis known in aqueous solution (Clore et al., 1987) and in crystal complex with carboxypeptidase A (Rees and Lipscomb, 1982). The27-residue globular core of PC1 isstabilized by three disulfide bridges and lacks regular secondary structures except for a short 5-residuehelix, positions 14-18. Nevertheless, PC1 shows a defined three-dimensional structure, being a good example of a minimal globular protein. Residues 35-39 form a C-terminal tail that protrudesfrom the globular core. The exact biological role of PC1 is unclear, although it is probably involved inplant defense againstfungalattackand 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 severaldifferentprotein families (Holm and Sander, 1993). PC1 can establishcomplexes with several carboxypeptidases inhibiting them in a strong competitive way with a K, in the nanomolar range. The best characterizedcomplex is that with bovine carboxypeptidaseA(CPA)(Rees and Lipscomb, 1982). In this complex, the C-terminal aminoacid tail of PC1 docks into the active siteof the enzyme, leading to a stopper-like inhibition mechanism. In the first stages of the binding, the C-terSome protease inhibitors are among the smallest globular minal residue of PC1 (GlY3’) is cleaved offbyCPA, and the proteins known. They havebiotechnological and pharmaceuti- carboxylate group of the previous residue (VaP8)makes a coorcal applications because of their involvement, together with dinate bond with the active site Zn”. According to the crystal proteases, in important processes such as peptide processing, structure, all residues in the PC1 C-terminal tail makecontact defense mechanisms, fertilization,carcinogenesis, trauma and with CPA residues in the protease-inhibitor complex except for inflammation, virus replication, and others(Ribbons and Brew, Gly35. The functional importance of some contact sites in the 1976; Ryan, 1989; Billings et al., 1989; Fritz et al., 1990; Hoc- binding of PC1 to CPA has been experimentally determined by man, 1992; Aviles, 1993). The complexes of proteases with their chemical modification and enzymatic studies (Hass et al.,1976; Haas and Ryan, 1980). So far, there have been no systematic of the com* This work has been supported by grants BI091-0477, BI092-0458, studies on the basisof the thermodynamic stability and IN90-0009 from the CICYT (Ministerio de Educacidn y Ciencia, plex and the contributions toit of specific residues and chemSpain). Thecosts of publication of this article were defrayed inpart by ical groupsof PCI. the payment of page charges. This article must therefore be hereby In two previous studies (Molina et al., 1992; Marino et a l . , marked “aduertisement”in accordancewith 18 U.S.C.Section 1734 1994), a synthetic gene encoding the isoform IIa of PC1 was solely to indicate this fact. constructed and expressed in Escherichia coli using the secre$ PFPI fellowship recipient of the Ministerio de Educacidn y Ciencia (Spain). Present address: Unitat de Bioquimica i Biologia Molecular, tion vector PIN-111-ompA-3, fused in frame to the OmpA signal 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 The abbreviations usedare: PCI, potato carboxypeptidaseinhibitor; Iberoamerica. CPA, carboxypeptidase A , HPLC, high performance liquid chromatogll Towhom correspondence should be addressed. Tel.: 34-3-581-1233; raphy; Kj, inhibition constant; wt, wild-type; dATPaS, deoxyadenosine Fax: 34-3-581-2011. 5’-a-thiotriphosphate.

Although the carboxypeptidaseA-potato carboxypeptidase inhibitor (CPA-PCI) complexis a well known example 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-foldhigher than that of wild-type PCI,indicating that Vals8is a key residue. Theinteractions of Vals with CPA residues contribute 5.4-5.7 kcal mol“ to theoverall stability of the CPAPC1 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, wereconstructed, 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 participate 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.

21467

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

21468

TABLEI Inhibition constants (KJ of wild-type and mutantrecombinant PCIs and Gibbs free energy of dissociation (AGdo) of the PCI-CPA complexes The wild-type and mutant forms of PC1 were obtained as recombinant proteins in E. coli and purified as described under “Experimental Procedures.” Inhibition constants were calculated according to Henderson (1972) in the case of PC1 wild type and mutants delG39, V38L, V381, and V38F and according tothe Lineweaver-Burk methodin thecase of PCIs V38A, V38G, and delV38G39. Several independent determinationsof the inhibition constant were made for each form of PCI. The dissociation free energy of the PCI-CPAcomplexes was calculatedaccording to the formula AG: = -RT In K, Complex

Inhibition constant, K, Interval

AG: Mean

0.9-2.1 3.1-4.3 65-95 0.74-0.9 26-55 11.4-14.7 1.7-2.5 0.9-2.1

x 10-9 x 10-9 x 10-9

x

loe6

x x IO‘$ x 10-9 x

Mean kcal mol”

M”

Wild-type PCI-CPA PCIdelG39-CPA PC1 V38A-CPA PC1 V38G-CPA PCIdelV38G39-CPA PC1 V38L-CPA PC1 V38I-CPA PC1 V38F-CPA

of PCI-CPA complexes

Interval

11.6-12.1 1.5 x 3.7 x 76 x 0.82 x 41 x 13.0 x 2.1 x 1.5 X

10-9 10-9 10-9

10“j

10-9 10-9 10-9

11.3-11.5 9.4-9.6 8.1-8.2 5.7-6.1 10.5-10.7 11.5-11.7 11.&12.1

11.8 11.4 9.5 8.1 5.9 10.6 11.6 11.9

The K, values of the PC1 wild type and mutantsdelG39, V38L, V381, and V38F for the inhibitionof bovine CPA were determinedaccording 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-phenylalaninewas used as a substrate at different concentrations. For each, several measurementsof varying PC1 concentration were made. Enzyme (bovine carboxypeptidase A) was 42.5 nM in all cases. Substrate hydrolysis was followed by A,,, measures for 2 min.Velocities were expressedas the slope ments made every 20 of the linear increase in absorbance and used to calculateK,. Several independent K, determinations were performed for wild-type and mutant PCIs. The average K, for each form of PC1 and Gibbs free energy EXPERIMENTAL PROCEDURES (AG,’) for dissociation of the PCI-CPA complexes were calculated. Chemicals a n d Enzymes-Enzymes were purchasedfrom Boehringer Computer Simulation and Graphics-Molecular graphics and simuMannheim and Pharmacia Biotech Inc. [ C ~ - ~ ~ S ] ~ Awas T Pobtained C~S lations were performedon a Crimson Elan,from Silicon Graphics. The from Amersham Corp. M13 sequencing kits were from Pharmacia (T7 structure of wild-type PC1 was taken from the x-ray structure of the sequencing kit). Site-directed mutagenesis was performed using the PCI-IIa isoform in the complex with CPA (Rees and Lipscomb, 1982), Amersham kit. adding the C-terminal residueGly. The structures of the PC1 mutants Cloning, Site-directed Mutagenesis, and Gene Expression-The con- and the corresponding complexes with CPA were modeled and visualstruction of a synthetic gene for PCI, its expression in E. coli, and a ized from the wild-type PC1 structure by means of the TURBO FRODO procedure to purify recombinantPC1 secreted into the culture medium program (Roussel and Cambillau, 1991). These structures were optihave been previously reported (Molina al., et 1992; Marino et al., 1994). mized by 5000 steps of steepest descent using the GROMOS package The synthetic gene for PC1 was cloned in the pINIII-ompA-3 vector under the noninertial solvent (NIS) force field (van Gunsteren and (Ghrayeb et al., 19841, fused in frame to the ompA signal sequence, Berendsen,1987).Theposition of themainchainatomswas congenerating thevector pIMAM3. Mutagenesis and sequencing were per- strained, assuming that conformational changes in mutants are small formed after cloning the XbaI-EcoRI fragment of pIMAM3, comprising and do not significantly perturb the structure of PC1 (see “Results”). Calculation of the volumes of the optimized structures were made using the PC1 gene and the OmpA signal peptide, in vectors M13mp18 and M13mp19 (Yanisch-Perron etal., 1985). The strain TG1 (Gibson, 1984) the GEPOL program (Pascual-Ahuir et al., 1987). was used as a host for propagation of MI3 derivatives. Site-directed RESULTS mutagenesis of the PC1 gene was done according to the method of Nakayame and Eckstein (1986). The designated deletions and point To test the role of Val38in the stabilityof the PCI-CPA commutations were delG39, delV38G39, V38G, V38A, V38L, V381, and plex, a series of site-directed mutants was created: delG39, V38F. Mutant PC1 genes were recloned in the PIN-111-ompA-3 vector. Sequence analyses were performed by the dideoxy method (Sanger et delV38G39, V38G, V38A, V38L, V381, and V38F. All of these mutants were expressed in E. coli as extracellular soluble proal., 1977). teins. They showed chromatographic behavior in ion-exchange The E.coli strain MC1061 (Casadaban and Cohen, 1987) carrying the pIMAM3 plasmids was used to produce wild-type and mutant recom- and reverse-phase HPLC identical to thatof wild type. This fact binant PCIs,as previously reported (Molina et al., 1992). Wild-type and suggests that allof them have the same overall conformation mutant inhibitors were found in the culture medium. Cultures were and that amino acid replacements do not alter the disulfide harvested after 24 h by centrifugation (10,000 x g, 20 mid, and the pairing and thefolding of PCI. supernatant (extracellular medium) was keptfor repurification. Allof the mutants showed competitive inhibitory activity Zmmunodetection-Polyclonal purified antibodies against PC1 were used to detect it in enzyme-linked immunosorbent assays during pro- toward CPA although withvery different K,. Table I shows the duction and purificationof wild-type and mutant forms of the inhibitor. K, values for CPAof all of the mutantforms of PC1 as well as the They were obtained as previously reported (Molina et al., 1992). AGdoof the complexes calculated according to theequation AG,” Purification and Characterization of Wild-type and Mutant PCIs= -RT In K,. This calculation is feasible because in the case of Wild-type and mutant forms of PC1 were purified from extracellular competitive inhibitors such as PC1 the inhibition constant is a medium of E. coli (pIMAM3) cultures by ion exchange on DEAE fast protein liquid chromatography and reverse phase on C18 HPLC as true dissociation constant for the enzyme-inhibitor complex previously reported (Molina al., et 1992). PC1 was detected by inhibitory (Todhunter, 1979; Palmer,1985). The K, value for the wild-type measurement assays, according to Hass and Ryan (1981), and by en- recombinant PC1 was 0.9-2.1 x lo-’ M, corresponding to a AG,” zyme-linked immunosorbent assay. The concentration of the purified for wild-type PCI-CPA complex of 11.6-12.1 kcal mol-’. This solutions of recombinant PCIs was determined from the A,,, of the final value differs from that derived by Rees and Lipscomb (1982),11 solution (PC1 extinction coefficient, E,,,, = 3.0) and also by Bradford assay usingwild-type PC1 as a standard. Both methods gave the same kcal mol-’, because they used for their calculation the first K, results. Molecular masses were confirmed by mass spectrometry in a 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 M Kratos Kompact MALDI 3 V2.0 spectrometer.

peptide-encoding sequence. The recombinant PC1 was found almost exclusively in the culturemedium, not in theperiplasmic space, as would be expected from OmpA signal peptide fusions. In the present work we report the analysis, by sitedirected mutagenesis, of the role of the PC1 residue Val38 and its side chain chemical groups in the stabilization of the CPAPC1 complex. Our results indicate that both the side chain and the main chainchemical groups of V a P make a major contribution to the energetics of the CPA-PC1 interaction.

lo-’

2 1469

Key Role of VaP8 in Stability of CPA-PCI Complex

(Hass and Ryan, 1981) corresponding to a AG: of 11.7-12.0 a kcal mol-l. PC1 W-CPA 11.6-12.1 Kcallmol The K, of the first mutantof the series,delG39, was 3.1-4.3 x lo-' M, only slightly higherthan that of wild-type PCI, 0.9-2.1 0.9-1.6 Kcallmol x lo-' M. This fact indicates that the last residue of the C-tailof 2-2.7 Kcallmol PC1 V U - C P A 10.5-10.7 Kcallmol PCI, Gly39, has no important role in the stabilization of its Whole side-chain contribution complex with CPA. In fact, a highly homologous inhibitor from 3.4-4 Kcallmol 1 tomato lacks the C-terminal glycine (Hass and Hermodson, PC1 VRRA-CPA 9.4-9.6 Kcallmol 1981). In contrast, the mutant PC1 with deletion of the last two L3-CH2 1 i.2-1.5 Kcallmol I residues of the C-tail,VaP8 and Glf9 (PCIdelV38G39), showed PC1 V38G-CPA f a dramatic decrease of inhibitory activity toward CPA (its K, 8.1-8.2 Kcallmol increased about lo4 times with respect tothat of PCIdelG39). The stability of the PCIdelV38G39-CPA complex, expressed as b AG:, decreased to 5.7-6.1 kcal mol" (see Table I). The overall G~Y Ala Val Leu Ile Phe contribution of VaP8t o the stabilityof the PCI-CPAcomplex can be estimated by comparing the AGdo obtained for the PCIdelV38G39-CPA complex with that of the PCIdelG39-CPA complex, because thedifferences in the respective K, values and stabilities of the complexes are attributable to the absenceof contribution limit the residueVal38.We can therefore estimate that V a P contributes 5.2-5.8 kcal"mo1" to the overall stability of the natural FIG.1. a, differences in dissociation free energies (AG:) of the comcomplex. This value represents about halfof the total stability plexes formed by CPAand the recombinant PC1 wild-type, V38L, V38A, of the complex, 11.6-12.1 kcal mol-l, confirming that VaP8 is a and V38G mutants. The energetic contributions, estimated from these differences, of each aliphatic group of the VaP side chain in the overall key residue for the inhibitory activity of PCI. stability of the PCI-CPA complex areindicated. b, schematic represenThe low inhibitory activity shown by PCIdelV38G39 tation of the side chain groups of the residue in position 38 in PC1 V38G, prompted us t o obtain the point mutants V38A and V38G to V38A, wild type, V38L, V381, and V38F. According to our hypothesis, estimate the contribution of the interactions established by the only aliphatic groups in positions and y establish hydrophobic interactions with atoms of the active site ofCPA in the protease-inhibitor different side chain groups of PC1 VaP8 with residues at the complex. Methyl groups in position 6, present in mutants V38L and CPA active site. The large increase in the inhibition constants V381, donot establish such interactions and thereforedo not contribute observed for both mutants (Table I) with respect to the wild to the stability of the PCI-CPA complex. type, from 0.9-2.1 nM to 65-95 nM and 740-920 nM, respectively, clearly indicates that the side chain aliphatic groupsof equivalent, they are likely t o contribute equally, 1-1.4 kcal Val38play an importantrole in the binding CPA. to The mutant mol", to the overall stability of the PCI-CPA complex. This V38G cannot establish with CPA those interactions involving value is similart o the contributionof the P-methylene of Val38 the three aliphatic side chain carbonsof VaP8. Therefore, the deduced above. These results suggest that hydrophobic the condifference in AG: between the complexes ofCPA with PC1 tribution of each methyVmethylene group of residue 38 to the V38G and with wild-type PCI, 3.4-4 kcal mol-', is an estima- AGdo of the PCI-CPA complex is the same, about 1-1.5 kcal tion of the hydrophobic contribution of these three aliphatic mol". carbons to the overall stability of the complex (Fig. la). As The mutants so far analyzed indicated that the side chain previously discussed, the total energetic contribution of Val38is hydrophobic contribution of the residue inposition 38 of PC1 is 5.2-5.8 kcal mol-'. The difference between this contribution very important for the stability of the protease-inhibitor comand the hydrophobic contribution of the side chain is 1.2-2.4 plex. In order to find out if it was possible t o obtain a better kcal mol-', which can be attributed to the other interactions inhibitor by increasingthis hydrophobicity, we constructed that Val38 establishes with CPA (two hydrogen bonds and a three more mutants: V38L, V381, and V38F. Surprisingly, the coordinate bond with the Zn2+atom of the enzyme (Rees and mutant V38L was less active than wild-type PC1 (the free enLipscomb, 1982). Estimations of the free energy of hydrogen ergy of the PC1 V38L-CPA complex is 10.5-10.7 kcal mol"). In bond formation, basedon studies with mutants, gave values of contrast, the mutant V38I presented a K, indistinguishable 0.5-2.0 kcal mol-' per hydrogen bond(Serrano et al., 1992). Our from that of wild-type PCI. This fact was unexpected because results are consistent with these estimations. both leucine and isoleucineside chainshave four methyl/ The second mutant, PC1 V38A, showed a free energy of dis- methylene groups, varying only in their positions. In order to sociation with CPA of 9.P9.6 kcal mol". The only difference explain these results, we formulated the hypothesis that the between mutants V38G and V38A is the side chain of residue stability of the PCI-CPA complex depends not on the overall 38 (a hydrogen atom and a methyl group, respectively) (Fig. side chain hydrophobicity of residue 38 of PC1 but only on the la). The difference in Kiand stability of the PCI-CPA complex additive hydrophobic contributions of the methyYmethylene between both mutantsis, therefore, attributable to thehydro- groups inpositions P and y (Fig. l b ) .According to this hypothphobic contribution of the side chain methyl group in position esis, the aliphatic groups in position 6 of residue 38 of PCI, 38 of PC1 V38A. This contribution canbe estimated as 1.2-1.5 present in mutants V38L and V381, would not make any conkcal mol-'. Because this methyl group is equivalent to the tribution to the stabilityof the PCI-CPA complex. methylene in position P of VaP8 in thewild-type PCI, it can be Leucine has a methylene in the P position and another in the the assumed that the contributionof the latter group to overall y position, whereas valine has one methylene in P and two stability of the wild-type PCI-CPA complex is also 1.2-1.5 kcal methyls iny. If the above hypothesis is right, the CPA complex mol-'. with V38L should havea AGdo1-1.5 kcal mol" lower than that The difference in AGd0 between the complexes of wild-type of the complex with wild-type PCI, because it loses one of the PCI-CPAand PC1 V38A-CPAis 2-2.7 kcal mol-', attributable t o methylene groups iny that contributed 1-1.5 kcal mol" to the the two y-methyl groups of Val3*, absent in the mutant PC1 binding. The difference found experimentally between the AG: V38A (Fig. 1, a and b). Becauseboth groups are sterically of the two complexes was 0.9-1.6 kcal mol" (Table I), inperfect

I1 1

I

Key Role of VaP8 Stability in

21470

of CPA-PCI Complex

agreement with the value predicted according to our hypothea sis. This hypothesis also allows us to explain the K, of the - 8.20 mutant V38I. Valine and isoleucine have the same numberof Ile methyurnethylene groups in the p and y positions of their side chains. Therefore, their complexes with CPA should have the same AGdo,and that isexactly what wasexperimentally found linhibifor] (Table I and Fig. 1). A computer simulation was performed to gain insight into Ala the thermodynamic basis of the bindingaffinities of the differP ent PC1 variants in residue38. The mutants in thecomplexes 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, 63.60 I 8.00 0.0 0.5 I .o 1.5 2.0 2.5 and an optimization of the structure by energy minimization Solvation Free Energy (kcal mol.') was performed to avoid nonallowed contacts of the mutated side chains. This approach assumes that the conformational b changes in mutant PCIs are small and do not significantly V38G 0 perturb positions of the main chain atoms of PC1 in the PCI4'00 CPA complex. First, the inhibitor establishes many contacts 3.00 ; with CPA (Rees and Lipscomb, 1982), and only a few of them 7F v38/ are directly affected by mutations ofVal38; but more impor2.00 tantly, all of the mutantsconserve a considerable (competitive) ?2 inhibitory power, particularly those carrying thebulkiest resi, -1.00 dues. 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 38is accommodated at the large active site cavity, as shown below. After -1.00 energy minimization the surface triangulation program GE0.0 20.0 50.0 80.0 110.0 POL (Pascual et al.,1987) wasused tocalculate the volumes of - AV'''' (nm3 X 1 03) wild-type and mutant PCIs and of their complexes with CPA in 2. a , total volumes of wild-type PC1 and mutants, alone (right) order to calculate the differences in volume between mutants andFIG. in complexes with CPA (left),uersus the solvation free energies of and their complexes. The excluded volume when forming the mutated residue 38 of PCI, according to Eisenberg and McLachlan was calculated according to the expression, complex, Vexel, (1986). b , increase of AG," of CPA-PC1 complexes versus the difference

'

8

;

F

1

l"'v

= vCpA + vP,I

- vCPA.pCI

(Eq. 1)

where V, is the volume of CPAalone, V,,,, is thevolume of PC1 alone, and V, is the volume of the complex. The mutationsof residue 38 only affected the hydrophobicity and volume of its side chain. Therefore, we assume thatmainly the entropic factors of the interactions between PC1 and CPA were perturbed. Computer graphic analysis of the energy-minimized complexes showed that theelectrostatic interactions and hydrogen bonds between the molecules were essentially maintained 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 tobecome buried, using the solvationfree 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 (notincluded 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 t o the wild-type PCI-CPA complex and the increase in theexcluded volume. We can calculate the change in dissociation energy of the complexes A(AG) relative t o the wildtype 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-

of excluded volumes. The slope of the linear regression (rn = 0.04) lies within the rangeof the predicted value derivedfrom Equation 2 ( K T p ) (see "Results"). V, excluded volume of the wild-type PCI-CPA complex; PC', 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 theincrease in theexcluded volume for the different PC1 variants (Fig. 2b). All of these results strongly support our hypothesis that any side chain aliphaticC6 in position 38 isnot buried in theactive 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 structuresof the different mutant inhibitor-CPA complexes in computer graphics helps us to visualize 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 mutantsV38I and V38L faces the watershell. 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 theS1 subsite of CPA. It can be seen that the8-methyl group of the mutant is directed backward to the PC1 core, specifically to TrpZ8of PCI, and hasno significant contacts with CPA. Therefore, it cannot contribute to the stability of the complex.

Key Role of Val38 Stability in

of CPA-PCI Complex

21471

FIG.3. Computergraphicsvisualization of the model-built mutantPC1 V38I interacting with CPA i n 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 andstick model, and the mutated Ile38 side chain is dark. Computer graphics representations were produced using the TURBO FRODO program.

w4*

The mutantV38F is worth a separate discussion. It showed with the Zn2+and with of the enzyme (interactions visua Ki indistinguishable from that of wild-type PC1 (Table I). This alized in thecrystal complex) but also can be attributed to the residue was not included in the graphs of Fig. 2 because, in loss of its side chain groups. Thus, 3.4-4 kcal mol"of the addition to theentropic effects, it shows a significant enthalpic PCI-CPA complex dissociation energy are attributable to the contribution ( P interactions) with nearby aromatic residues. hydrophobic interactions of the Val3' side chainwith residues of This is why the hypothesis we used to explain the results ob- CPA, the contribution of each of the methyllmethylene groups tained for the other mutantscannot be used in thiscase. Phen- of this side chain being1-1.5 kcal mol". This estimate fits well ylalanine has justone aliphatic Cpplus an aromaticring. The into the range of values reportedelsewhere for the contribution contribution of this ring to the stability of the PC1 V38F-CPA of aliphatic groups to the stability of proteins (Kellis et al., complex can be estimated as 2-2.7 kcal mol-', that is the dif- 1989; Matsumara et al., 1989; Pace, 1992). The importance of ference in A G: between CPA complexes with PC1 V38A and Val3' in the binding of PC1 to CPA is probably related to the with PC1 V38F. Again, visualization by computergraphics important role of the subsiteslocally involved (P1 in theinhibhelpstounderstandthe experimental results. Computer itor and S1 in theenzyme). Both subsites are essential in the graphics analysis shows that the Phe3' phenyl group of the recognition, binding, and catalysis of oligopeptide substrates mutant does not seem to alter the structure and stability of the and inhibitors by CPA (Abramowitz et al., 1967; Christianson complex because it lies between the two nearby aromatic side and Lipscomb, 1989). In this respect, it is worth mentioning chains of of CPAand of Trp2' of PCI, the three rings being that theS1 subsite shows the higher degree of stereospecificity kept oriented in appropriateangles to make favorable P inter- between the enzyme subsites. This is the region in which the actions. Our estimation of the aromatic ring contribution, 2-2.7 substrate is submitted to a torsional strain thatfacilitates cakcal mol-', is in line with the reported range for the energetic talysis (Nakagawa and Umeyama, 1978). contribution of interactions between two aromatic rings in the The replacement of the Val3' side chain by shorter or longer stability of proteins (Hunter et al., 1991). apolar ones in mutantPCIs affects the inhibition constants in a trend thatsuggests that thisside chainshould have a precise DISCUSSION size (ie. that of Val) to efficiently bind to CPA. The analysisof Previous knowledge of the relative or quantitative contribu- the series of mutants in thisresidue shows that the stability of tion of PC1 residues to theinhibition of carboxypeptidase A was the complexes with CPA depends not on the overall side chain limited. It was based on chemical modification and enzymatic hydrophobicity of residue 38 but on the hydrophobic contribustudies (Hass et al., 1976; Hass and Ryan, 1980) and on the tions of the methyllmethylene groups in positions p and y. analysis of contact areas in the crystal structure of the complex Computer simulation of the variation of the total volumes of (Rees and Lipscomb, 1982). These studies attributed thebind- PC1 and complexes versus solvation free energies or versus the ing andinhibitory ability of PC1 to the primary and secondary experimental changes in stability, together with the computer contact regions of the inhibitor without a deeper appraisal of graphics inspection of the interactions at the S1 subsite of CPA, their contributions. They also attributed a minor role to GlY9. corroborates our conclusions. This indicates that side chain Although this generalview is still valid, from the present study groups in position 6 of residue 38 would not contribute to the we can conclude that a single residue of PCI, Val3', is a key stability of the CPA-PC1 complex because they would be oriresidue because it contributes as much as the rest to the bind- ented out of the narrow passage of the CPA S1 subsite, not ing and inhibition. Thus, 4 5 4 8 % of the free energy of the buried in theactive center of the enzyme. binding, 5.2-5.8 kcal mol-' over 11.6-12.1 kcal mol-', is attribIt has been reported that it is difficult to formulate simple utable to thisresidue. This isa relevant resultconsidering that general correlations between dissociation energies of proteinthe other 9 residues of PC1 primary and secondary contact protein complexes and interactions in contact surfacesbecause regions have been shown to establishdefined contacts with the of the fact that the energetic contributions of certain kinds of enzyme in its crystal structure. interactions ( i e . hydrogen bonds and hydrophobic interactions) Our site-directed mutagenesis studies also show that the are not isotropic but are dependent on the geometry and envidecrease in binding energyafter Val3' removal cannot be exclu- ronment surrounding the interacting groups (Janin and Chosively attributed to the loss of the interactionsof its carboxylate thia, 1990; Walls and Sternberg, 1992). Nevertheless, in par-

w4'

21472

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

Henderson, P. J. F. (1972)Biochem. J . 127,321-333 Hocman, G. (1992)Znt. J. Biochern. 24, 1365-1375 Holm, L., and Sander, C. (1993)J. Mol. B i d . 233, 123-138 Hunter, C., Singh, J., and Thornton, J. M. (1991)J. Mol. B i d . 218, 837-846 Janin, J.,and Chothia, C. (1990)J. Bid. Chem. 265,16027-16030 Kellis, J. T., Nyberg, K., and Fersht,A. R. (1989)Biochemistry 28,491-922 Marino, C., Molina, M. A,, Canals, F., Aviles, F. X., and Querol, E. (1994)Appl. Microbiol. Biotechnol. 41, 632-637 Matsumura, M., Wozniak, J. A., Dao-Pin, S., and Matthews, B. W. (1989)J. Biol. Chem. 264,16059-16066 Molina, M. A,, AvilBs, F. X., and Querol, E.(1992)Gene (Amst. 116, 129-138 Nakagawa, S., and Umeyama, H. (1978)J. Am. Chem. Soc. 100,7716-7725 Nakayame, K. L., and Eckstein, F. (1986)Nucleic Acids Res. 14,9679-9698 Acknowledgment-We are grateful to M. Lockwood for revision of the Pace, C. N. (1992)J. Mol. Biol. 226, 29-35 manuscript. Palmer, T. (1985)Understanding Enzymes, 2nd Ed., pp. 145-147,Ellis Horwood Ltd., Chichester, U.K. Pascual-Ahuir, J. L., Silla, E., 'Ibmasi, J., and Bonacorsi, R. (1987)J. Comput. REFERENCES Chem. 8,778-787 Abramowitz, N., and Schechter, I. (1974)Isr: J . Chem. 12,543-555 Rees, D. C., and Lipscomb, W. N. (1982)J . Mol. Biol. 160, 475498 AvilBs, F. X.(ed) (1993)Innovations in Proteases and Their Inhibitors, Walter de Ribbons, D. W.,and Brew, K. (eds) (1976)Proteolysis and Physiological Regulation, Gruyter and Co., Berlin Academic Press, New York Billings, P. C., Morrow,A. R., Ryan, C. A., and Kennedy,A. C. (1989)Carcinogenesis Richmond, T. J. (1984)J. Mol. Biol. 178, 63-89 (Berlin) 10,687-691 Roussel, A,, Inisan, A. G., and Knoops-Mouthy,E. (1994)TURBO FRODO fuersion Casadaban, M. J., and Cohen, S. N. (1980)J. Mol. Biol. 138,179-207 5 . 0 ~Manual, ) BIOGRAPHICS, Technopole de Chateaux-Gombert, Marseille, Christianson, D. W., and Lipscomb, W. L. (1989)Acc.Chem. Res. 22, 62-69 France Clore, G. M., Groneborn, A. M.,Nilges, M., and Ryan,C. A. (1987)Biochemistry 26, (1989) Bioessays 10,20-24 Ryan, C. A. 8012-8023 Ryan, C. A,, Hass, G. M., and Kuhn, R. W. (1974)J. Bid. Chem. 249,5495-5499 Eisenberg, D., and McLachlan, A. D. (1986)Nature 319, 199-203 Fritz, H., Schmidt, I., and Turk,V. (1990)B i d . Chem. Hoppe-Seyler371, 111-118 Sanger, F., Nicklen, S., and Coulson, A. R. (1977)Proc. Natl. Acad.Sei. U. S. A. 74, 5463-5467 Ghrayeb, J., Kimura, H., Takahora, M., Hsiang, H., Masui, Y., and Inouye, M. Serrano, L., Kellis, J. T., Jr., Cann,P., Matouschek, A., and Fersht,A. R. (1992)J . (1984)EMBO J. 3,2437-2442 Mol. B i d . 224, 783-804 Gibson, T. J. (1984)Ph.D. thesis, Cambridge University Todhunter, J. A. (1979)Methods Enzymol. 63,391393 Hass, G. M., and Hermodson, M. J. (1981)Biochemistry 20,22562260 Hass, G. M., and Ryan, C. A. (1980)Biochem. Biophys. Res. Comrnun. 97, 1481- van Gunsteren, W. F., and Berendsen, H. J. C. (1987)in Groningen Molecular Simulation (GROMOS) Library Manual(van Gunsteren,W. F. and Berendsen, 1486 H. J. C., eds)BIOMOS, Groningen, The Netherlands Hass, G. M., and Ryan, C. A. (1981)Methods Enzymol. 80,778-791 Haas, G. M., Ako, H., Grahn, D. G., and Neurath, H. (1976)Biochemistry 15, Walls, P. H., and Sternberg, M. J. E. (1992)J. Mol. Biol. 228, 277-297 Yanisch-Perron, C., Vieira, J., and Messing, J. (1985)Gene (Amst.)33, 103-119 93-100

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 improvement of the properties of proteins and protein-ligand complexes.