Conformational predictive studies on the activation seoment of pancreatic procarboxypeptidases

Vol. 149, No. 2, 1987 December 16, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 729-734

CONFORMATIONAL PREDICTIVE STUDIES ON THE ACTIVATION SEBMENT OF PANCREATIC PROCARBOXYPEPTIDASES M. Vilanova’ , F.X.AvilC’*,

J. Vandrell’ and E. Mar&$

’ Departament de Bioquimica and lnstitut da Biologia Fonemental, Universitat Autonoma.08093 Belleterra (Barcelona). Spain 28ervicio da Enckcrinologia, Centro Rambny Cejal. Carretera daColmenar Km. 9.1. 28034 Madrid. Spain Received

October

28,

1987

Little is knownabouttheconformationandevolutionaryorigin of the activationsegment of pancreatic procarboxypeptidases. Analysisof the sequence andsecondarystructure propensitiesof thesepropeptida segments indicatethat theycontaintwo regionsstructurally relatedto the &+-binding sitasof the EF-hand protein family. This proposed homology couldexplainhow(andwhy) carboxypeptidaaes developed suchlong (94 residues)activationpeptides. B 1987 Academic press, kc. The acceptedmodelfor the evolutionary pathwayfollowedby the propeptidaregionsin digestive proproteases suggests that theseregionshaveevolvedandhavebeen&dadto tha proteinafter tha appearance of the enzymaticregions( 1). This hypothesishasbeendeduced largely asa result of comparativesequence analysis of different and evolutionary distant serine-proproteineses (1,2). The lack of sequence information precludessimilar comparisonsin the caseof another important family of digestive proprotaases, that of pancreaticprocarboxypeptidesas. In pancreaticprocarboxypeptidases, the propeptideregionis locatedat the N-terminusandcomprises aboutonequarterof the approximately400 residuesof the wholesequence (3,4,5). Wehaveshownthat in the A form of the porcineproanzyme,the propeptideconstitutesa globular structural domain(6,7). Moreover,whenisolated,it behwesasa strongcompetitiveinhibitor of carboxypeptideses A(4). We haverecently reportedthe completeprimary structure of the 94 residueactivationsegment of porcine procarboxypeptidesaA (8) and the 38 N-terminal homologousresidue-aof porcine prccarboxypeptidaseB (5). Comparisonof these regions with the equivalent sequencein rat procarboxypeptidase A, deducedfrom c-DNAby Quintoet al. (91, showsthat the structure of these activationsegmentsis largely preserved(8). At present,the evolutionarycloseness of theseactivation regionsandthe lackof informationaboutthe structure-function relationshipsin themdoesnotallowUs to confirm that thq follow the abovementionedcommonevolutionary pathww for propeptidaregionsin digestiveproproteinases. *To whomcorrespondence shouldbe addressed. Abbreviation%asA,asB: activationsegments of procarboxypeptideses A andB; EFCaBP : EF-hand &’ bindingprotein.

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0006-291X187 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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However,a closeanalysisof theabovereportedsequences of procarboxypeptidases suggests that their activationsegments containtworegionsstructurally andevolutionarilyrelatedto the &+-binding proteins of the EF-handfamily (calmodulin,troponin C, parvalbumin,..etc.), regionswhich have a potential capability to bind this metal. This unexpectedfinding and its possiblefunctional and evolutionary implications8re discussed andsubstantiated in thepresentpaper. RESULTS ANDDISCUSSION A detailedanalysisof the alternationof residuesof different physico-chemical characteralongthe presently knownsequences of the activationsagment of procarboxypeptidases indicatesthat thesesegments containtwo regionswhich satisfy several fundamentalrequirementsfor the &+-binding regionsin EFCaBPs. Accordingto different authors( 10,l I ,121 the &?-binding regionsin the EFCaBPs are formed by about29 residueswhich fold into two lateral a-helicesbridgedby a central loopof 12 residues,as shownin Figure 1. TheG?’ bindsto the loopby coordinationwith six oxygensfrom different residueside chains(namedX, Y, Z, -Y, -X, and-Z in the Figure), or from a few main chaincarbonylsor water molecules,in substitution. Followingthe above rules, the putative &+-binding regions in the activation segmentsof procarboxypeptidases are lccatedin residues 3-34 and55-84 in procarboxypeptidaseA (asA- and&-II,

loop

Nt-helix

A01

123456709 n EFCaBP Consensus Sequence

asA

I rat

asA I

Ct-helix

2345678901

rrrcrrrrn

rrrr,

nn

n

u&k

E@&

E@

X Y Z -Y -x

rrrrrrrr,

-z

2345

6709

n

nn

n

(3-34)

pig

(3-34)

D@JV

GHOQL

RO

Qv@DQAGZ~IV~K~K~)

QE

8SB

I pig

(6-37)

HOE

dK@F

H@

@V@DaN@I@Eka

@AH&lDG

BSA

II

rat

t55-&t4)

SaOS@KAFa

$HGlSYO)I

asA

II

pig

(55-84)

S(j)OA@KVFo

&-lGl

l?Y@l

~8

MI

Ea

@OLGJDDEE

MI

E&)

@XJLmDEE

EHL oQ

Figure I .- Canparison between the proposed calciumbindingregions of theactivationsegments of differentpr~boxypeptideses end a cunsensussquence for the EF-hand &?+ binding sites At the top, the figureshows a schematic representtiion of thelatter; n = hydraphobic residue; X,Y,Z,-Y,-X,-Z= residues that cmrdinate I$+. The encircled residues mnform the basic rules for the formation of typical EF-hand

C%?+ binding sites.

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either in rat or in porcine procarboxypeptidase A, and 5-37

(asB-I)

in porcine

procarboxypeptidase B (the region sequencedso far). To obtain a proper fit,a few artificial deletions must be introduced into each of the selected regions (according to a procedure already adoptedby the above authors), es shown in Figure 1. Thqhydrophobicresidues are mostlyfoundin positions Z-5-6-9 of N-terminal and C-terminal regions in each formulated Q?-binding

site in the activation segments -en

alternation which is usual in EFCaBPs. More important still, five to six oxygen-containing residues in a-4-I and asB-I arein the proper locationtocoordinateQ?+ in the proposedcentral loop.The ad-11 loop contains only three to four residues of this kind but the other positions required to coordinate Ca2+ may be supplied by the main chain carbonyl ( 10). Similar cases are found in typical EFCaBPs, such es sites I in rabbit skeletal troponin C (in Z position),cardiactroponin

C ( in X and Y positions),and porcine intestinal

calcium binding protein (in X position). To obtain a more detailed comparison, a “consensus” sequencefor the &?+ binding sites in typical EFCaBPswas established by selecting the residues most frequently found in each position of these siteson the basis of the Gariepy and Ho&&s study of 30 of these sites in different EFCaBPs( 11). This consensus EFCaBPsequence is also shown in Figure 1. Clearly this sequencesatisfiesall the mentioned fundamental requirements for EFCaBPs. The comparison could begin with the best preserved residue in EFCaBPswhich is glutamic acid in -2 position since it strongly supports the formation of the helical turn which forms the C-terminal part of the loop ( IO,1 1). This residue isalso foundatthe same position in W-1 (rat and porcine) and in asB-I, but is an aspartic acid in as+II. The next residue, position 2 at the C-terminal helix, is also very well preserved in EFCaBPs being a leucine in the sites I and III (or evolutionarily related, es site II in parvalbumin) or a phenylalanine in sites II and IV (or evolutionarily releted, as site III in parvalbumin) (10,ll).

For this position again we found a higher coincidence in asA- (rat

and porcine) and asB-I,

which contain a leucine,than in asA- which contains avaline. It isalsu worth mentioning that isoleucine is found at position 7 of the loop in ad-11 (rat and porcine) and in asB-I, es in most EFCaBPs ( I I). A valineoccupiesthesame position in &-I

(rat and porcine) but is alsoa relatively commonresidue in this

p1aceinEFCaBPs(10,11,12). One of the important failures in the fitting occurs in position 6 in the loop, usually occupied by glycine in EFCaBPs(whereit

playsan importantconformetional role),whereas

neither this residue noran

equivalent residue is found in this position in the activation segment of procarboxypeptidssas. However, similar failures in the fitting can also be found in distorted or in defunct sites of some EFCaBPs, such es sites I ofSlOCa,SlOOb, intestinal calcium binding protein,andsites II and III of rabbit skeletal muscle alkali light chain (10). Another important failure would appear to exists at position X which is usually occupied by an Asx residue in EFCaBPs ( 12,13).As pointedout by Herzberg andJames (13),this residue plays an important role in the loop conformation establishing an Asx turn by hydrophobic bonding to the backbone NH of residue at position Y. However, the same role may be played by a Ser residue ( 131, which is the kindofresidue

found in ad-1 (rat and porcine) as well as in other unusual EFCaBPs,such as bovine

SlOOaandSlOOb (iO,ll). inthisposition.

Inanycase, a better fit is displayedfor mB-I which presentsan asparagine

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I

!

0.3 0.2

;; -200 -300 400 -500

-0.1 -0.2 -0.3

10

20

30

40

50

Residue

-0.3J

: 10

! 20

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-0.31

!

10

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!

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! 40

40 Residue

70

80

90

100

: 70

: 80

! 90

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: 70

: 00

: 90

100

20

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40 Residue

50 00 number

70

00

90

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! 40 Residue

! ! SO 60 number

! 70

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I 100

number

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number

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70

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FiQUfe 2.- %YJCtUrd probability profiles for porcine a& (+-+) and for the hybrid, EF@@ wnsensus sequence() derived according the following methods: (A) and (C) Chou-Fasman for a-helix and reverse turn; (6) end (D) Vernier et al. for a-helix and reverse turn; (E) Hopp-Woods for lqdrophilicity; (F) Sweet-Eisenberg for hydrophobicity.

It is well knownthat conformationis better preservedthansequence in the evolutionof homologous proteins( 14,151.In order to checkthe possiblepreservationof basicconformational traits of EFCaBPs in the putative&+-binding sitesof the activationsegments of procarboxypeptidases, a comparativeanalysis of both kind of proteinswas carried out by different sequence-besed predictivemethodsChouandFesman ( 161, 9arnier et al. ( 171, SweetandEisenberg( 18) andHoopandWoods( 19). Accordingto different authors( t8,20) the comparativeuseof predtctivetechniques to establishhomologles betweenproteinsis more reliable than prediction itself since tong range interactionsare often equivalentin homologous proteins. We selectedthe ‘consensus*’ sequence of EFCaBPs es beingrepresentativeof theseproteinsto carry out the abovecomparison with predictive methods. Moreover,to avoidthe effect of the endsonthese predictive methods, a “hybrid” proteinwas built by substitutingthis ‘consensus” sequence for eachof the two putative bindingsitesin porcineasA.Thestructural predictionwasthereforecarriedout on the whole EF-consensus/porcine asA‘hybrid” sequence. The structural probability profiles of porcine asA and the “hybrid” EF-consensus/porcine asA sequence obtainedusingdifferent predictivemethods are shownin Figure2. Usingthe secondery structure predictive

methods of Chou and Fesman and of Qsrnier

732

et al., the structural

motif a-helix/reverse

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Table I .- Qsrrrelation coefficients between the activation segmentof porci ry procarboxypeptidase A (asA) and a EF-&?+ binding consensussequence*

asA P,-Chou-Fasman Pt-Chou-Fasman P,-Qarnier et al. P -6arnier et al. Hf-Hopp-WC& Hf-Sweet-Eisenberg

asA-

(l-100)

(3-34)

0.6602 0.7294 0.7583 0.6587 0.5694 0.5397

0.2803 0.4398 0.5855 0.3933 0.4356 0.3796

USA-II (55-84)

0.4186 0.3170 0.8710 0.8255 0.242 I 0.2769

(a) Whole asA is correlated with the EF-consensus / asA hybrid sequence. asA- and a%11 are correlated with the EF-consensus sequenceshown in Figure 1, Seetext for more details.

turn/a-helix clearly appearsin each&+-binding site in the consensus hybrid sequence.This is the structural motif generallyfoundin typical EFCaBPs ( IO,1 1,131. Thesamemotif is clearly apparentin the asA-11site within porcineprocarboxypeptidase Abut in a somewhat distortedform in theasA- site. In this site the probability for a-helix in the N-terminal regionis relatively low andits maximumis displaced towardsthe centerof the site wherea probability maximumfor reverseturn exists. It Is alsoworth noting the relative low valuesfor the peakmaximums of reverseturn probability in the centerof asA- and&-II comparedwith the correspondingvalues in the “hybrid” EFCaBPsequence model. In spite of these differencesbothkindsof proteinspresenta similar structural pattern. It is important to indicatethat the abovedifferencestend to disappear whenthe comparison (not shown)is madewith thefirst putative $+-binding site in asB(asB-I). Theconformational similaritiesbetweenasAandthe hybrid EFCaBP/asA sequence are confirmedwhentheir hydropathyprofileserecompared asshownin Fig2E endF. Further evidenceof conformationalhomologies betweenthe asApieceandEFCaBPswas found when the structural corrrelation coefficientsbetweenthe wholeas4andthe ‘hybrid” consensus sequence/asA sequence were obtainedfrom the abovementioned structural predictiveprofiles, followingthe procedure established by Pongorand&ala/ (201, asshownin Table1. Correlationcoefficientsmuchhigherthan 0.2 are obtainedfor all the different predictive methods,thus providingclear evidenceof homology(20). Similar resultsandconclusions are obtainedwhenanalysesare limited to the two putative &+ binding sitesof asAandthewnsensus sequence (seealsoTable1I. In the light of the above results we suggestthat the activation segmentof pancreatic procarboxypeptidasas wntains two putative EF-hand@-binding sites, at least in rat and porcine proenzymes.The recently publishedpartial sequence of as4from bovine prccarboxypeptidasa A (2 1) wnfirms our analysis. The strong effect of CC?+on the proteolytic activation of pancreatic procarboxypeptidases -a reportedfact for bovine(22), cbgfish(23) andlungfish(24) proenzymes -could berelatedto the bindingof this cationto the abovesites.However,thosesitesin porcineproenzymes should present a distorted conformationand affected ability to bind tit with regard to the normal EF-&+-binding sites, since th lack important residuesin their sequence.Conformationaland physiw-chemicalstud&sare in progressto confirmthesastructural traits. 733

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Th8 evidence w8 ha!‘8 found of the existence of relationships between EFCaBP 8nd th8 activetion regions of procarboxypeptidases allows us to formulate a preliminary hypothesis as to the evolutionary origin of these regions. Thus, these regions would not be the result of a simple and short extension of the geneof the protease, as hes been proposed in the case of serineproproteinases ( 1,251, but the result of the fusion of this gene with the gene of 8n EFCaBP or 8 related protein. This would explain why pancreatic procarboxypeptidases developed such a long (and biologically expensive) activation region, while it has been shown that short peptide regions are enough to inactivate these exoproteases (26). Analysis of the structure of the genesof these proenzymes will be required to test this hypothesis. ACKN-S

This work has been supported by grant 0385184 from the Comisibn Asesora ds Investigaci6n Cientificey TCnica (CAICYT), Ministerio de Educacibn,Spain. REFERENCES ( 1) (2) (3) (4) (5) (6)

Neurath, H. ( 1984) Science 224,350-357. Neurath, H. and Walsh, K.A. ( 1976) Proc. Natl. Amd. Sci. USA 73,3825-3832. Martinez, M.C.,Avil&, F.X., SanSagundo,B.,endCuchillo,C. M. (1981) Biochem.J. 197, 141-147. San Segundo, B., M8rtine2, M.C., Vilanova, M., Cuchillo, CM. and Avilb, F.X. (1982) Biochim. Biophys. Act8 707,74-80. Avilb, F.X., Vendrell, J., Burgos, F.X., Soriano, F., and M&-&z, E. ( 1985) B&hem. Biophys. Res. Comm. 130, 97-103. AvijbilbX., San Segundo,B., Vilanova, M., Cuchillo, C.M. and Turner, C. ( 1982) FEBS. Lett. 149,

Vilanova, A., Burgos, F.J., Cuchillo, C. M., andAvilb, F.X. ( 1985) FEBS Lett. 19 1 273-277. Vendrell, J., Avilb, F. X., &es&, E., San Segundo,B., Soriano, F., 8nd M&&z, i.( 1986) Biochem. Biophys. Res. Comm. 141, 517-523. (9) &into, C., Quirogs,M., Swain,W.F., Nikovits, W.C.,Standring,D.N.,Pictet, R.L.,Valenzuela,P. and Rutter, W.J. ( 1982) Proc. N8tl.Acad.Sci. USA79, 3 l-35. ( 10) Kretsinger,R.H.( 1980) CRCCritical Reviewsin Biochemistry8, 119- 174. ( 11) Bari@y,J. andHodges, R.S. ( 1983) FEBSL&t. 160, l-6. ( 12) Szebenyi,D.M.E.andMoffat, K. ( 1986) J. Biol. Chem.261,8761-8777. ( 13) Herzberg,0. andJames,M.N.B.( 1985) Biochemistry24,5298-5302. (14)Schultz, Q.E.8ndSchirmer, R.H. (1979) in Principles of Protein Structure (C.R. Cantor, ed.), Springer-Verlag,NewYork. ( 15) Doolittle,R.F.( 1981) Science2 14, 149- 159. ( 16) Chou,P.Y.andFasman, B.D.( 1978) Adv. Enzymol.47, 45- 141. ( 17) Qarnier,J., Osguthorpe, D.J.endRobson, B. ( 1978) J. Mol. Biol. 120, 97- 120. ( 18) Sweet,R.H.andEisenberg, D. ( 1983)J. Mol. Biol. 17 1 I 479-488. (l9)H~up,T.P.8ndWa&,K.R.(198l)Proc.N8tl.A&Sci.USA 78,3824-3828. (20) Pongor,S.andSzalay,A.A.( 1985) Proc. Natl.Acad.Sci. USA82,366-370. (2 1) Chspus, C., Kerf8le&B., Foglizo, E.andBonicelJ. ( 1987) Eur.J. Biochem.168,X-M (22) Uren,J. R. andNeurath,H. ( 1972) BiochemistryI 1, 4483-4492, (23) Lacko,A.8. andNeurath,H.( 1970) Biochemistry9,4680-4690. (24) Reeck,O.R.andNeurath,H. ( 1972) Biochemistry11 I 3947-3955. (25) N8ur8th,H. ( 1986) J. Cell.Bicchem.32,35-49. (26) Rees,D.C.andLipscomb,W.N.( 1982) J. Mol.Biol. 160, 475-498. (7) (8)

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