Conformational studies of an undecapeptide reproducing the consensus sequence around the cleavage...

Conformational Studies of an Undecapeptide Reproducing the Consensus Sequence Around the Cleavage Site of the RXVRG Endoprotease from Xenopus laevis Skin

DANIEL BARON, ',* ANNE-MARIE LESENEY,' FRANCOIS-REGIS CHALAOUX,' and JACQUES RIAND'

'LASIR, CNRS, 2 rue Henry Dunant, 94320 Thiais; and 2Biochimie des Signaux Rkgulateurs Cellulaires et Molkculaires, Universitk Paris VI et CNRS URA 1682, 96 boulevard Raspail, 75006 Paris, France

SYNOPSIS

Two synthetic fragments, corresponding to the 4-9 and 4-14 sequences of a tetradecapeptide used as a model to test the RXVRG-endoprotease activity from Xenopus laeuis skin, have been studied by two-dimensional nmr spectroscopies, correlated spectroscopy, and nuclear Overhauser effect (NOE) spectroscopy. Both peptides wore the 5-9 consensus sequence found in several hormonal precursors. The nmr data for the 4-9 hexapeptide did not indicate any particular organization, either in water or in dimethylsulfoxide (DMSO) , whereas, the 4-14 undecapeptide, a substrate for the RXVRG endoprotease, showed, in DMSO solution, significant trends of structural organization involving the amino acids pertaining to the consensus domain.

From variations of integrated NOE peaks with temperature, the apparent interproton correlation times T, were estimated and the maxima observed with Va17, the central residue in the consensus sequence. A defined tertiary structure in that domain was also supported by medium- and long-range NOES between Asp6 and Arg8, Glu4 and Gly9, and by the likely involvement of Arg8 and Gly9 NHs in intramolecular hydrogen bonds. Most of these observations could be rationalized by an equilibrium between a 5-8 @-turn and a 9 + 4 H- bonded loop.

The predominance of one rotamer for the Ca-Cp bond was established in four residues. Finally, the average @ and \k angles were derived from two models taking, or not, into account variations in the correlation times along the sequence. This allowed us to discuss the artefacts generated by using an average correlation time through the whok molecule. 0 1994 John Wiley & Sons, Inc.

I NTRO D UCTlO N

Endoproteolytic cleavage of precursor proteins, which usually occurs at pair basic sites, or occasion- ally at mono- or oligobasic sites, is one of the key steps in the production of bioactive peptides and proteins.' Endoproteases are responsible for these proteolytic conversions, but only a few of these have been isolated.'-' Studies on the probable mechanism of action of these enzymes show clearly tha t they not only recognize basic amino acids, but also certain

Biopolymers, Vol. 34, 1419-1431 (1994) 0 1994 John Wiley & Sons, Inc. CCC 0006-3525/94/101419-13

* To whom correspondence should be addressed.

conformational arrangements in the surrounding of amino acids situated at the cleavage ~ i t e . ~ " - ~ ' In ad- dition, specialized peptide sequence, RX ( K or R ) R or RXXR, identified at the cleavage site of several proteins, especially proreceptor, prohormonal, and proviral proteins, 13,14 seems to influence greatly the cleavage by processing endoproteases of the furin fami1y.l4-l6

Xenopus laeuis, as numerous amphibians, syn- thesizes a variety of biologically active peptides in its kin.^^^'' Nucleotide probes, designed with the active peptide sequences, allowed the isolation of mRNA coding for their precursors. Sequence analysis evidenced numerous precursors showing a high degree in sequence homology.1g~22 None of these

1419

1420 BARON ET AL.

precursors has been isolated to date; however, their nucleotide sequences show a particular organization, with a highly conserved sequence RXVRG, situated at the N-terminus of amphiphylic peptides (21-30 residues long) .6 These peptides, beginning by a Gly residue, have been identified in the X. laeuis exudate 23 and shown to possess antibacterian prop- ert ie~.’~ The high conservation, through the different precursors, of the pentapeptide situated at the cleavage site, suggests the existence of a functional constraint susceptible to allow the cleavage by the endoprotease implicated in the post traductional maturation.

A tetradecapeptide was designed from the 20 cleavage sites identified in the X. laevis precursors, and used as a probe to look for the endoprotease implicated in the maturation process.6 This tetradecapeptide possesses the consensus fragment RXVRG in which X is Asp. Six of the other amino acids were chosen because of the high frequency of their appearance in the corresponding domains of the precursors from X . laevis (over 50%): Aspl, Asp3, Glu4, Serl2, Phel3, and Leul4. The 10th po- sition is occupied by an hydrophobic residue for which the higher frequency observed is Phe before Ile and Leu. At last, residues Val2 and Alall correspond to the most frequent amino acid flanking Aspl and PhelO. Therefore, the retained tetradecapeptide was HDVDERDVRGFASFLNH, . It must be underlined that this sequence corresponds to one of the caerulein precursor cleavage site, except for the Asp6, which replaced an Asn residue in the na- tive sequence. Using this synthetic tetradecapeptide as a probe, an endoprotease, which selectively hy- drolyzes the Arg-Gly peptide bond of the highly conserved motif in the sequence, was isolated from the exudate of the X. laevis skin.6 This processing enzyme has been called “RXVRG” endoprotease. It behaves as a metalloendoprotease and exhibits some sensitivity to serine protease inhibitors. The enzyme, tested for activity on different peptides derived from the model tetradecapeptide, by substitution of either one or the other amino acid from the consensus sequence, appears to require the complete sequence to be fully active.6

In order to obtain some precision on the tertiary structure of the peptide and clarify the importance of the different residues in the architecture of the highly specific processing domain, we performed nmr studies both on the tetradecapeptide and some of its derivatives and fragments. The precursor consensus sequence is surrounded by a domain with a high content in Asp and Glu at the amino side and a highly hydrophobic region at the carboxyl side.

Therefore, it was important to define the role of each domain in the final structure. This paper reports on the results obtained in the study of two fragments containing the consensus sequence Arg5-Gly9: the hexapeptide 4-9 and the undecapeptide 4-14, this last peptide only being a substrate for the enzyme (A. M. Leseney, unpublished results). Scalar coupling constant J have been determined using the fine structure of cross peaks in the correlated spectroscopy (COSY) diagram ( JNa is useful for the @ angle and JaB for CaCP rotamers of the side chains). Nuclear Overhauser effects (NOE) have been calculated using an analysis of the overlaps in the NOE spectroscopy (NOESY) diagrams, followed by nor- malizations of the correlations allowing a realistic estimation of the distance restraints in these struc- t u r e ~ . ~ ~

These peptides were mainly studied in dimethylsulfoxide (DMSO-4) since the solubility in water of the undecapeptide was too low, probably due to the presence of four hydrophobic residues. In fact, protein ligand association generally involves both lipophylic and polar moieties and, according to Kes- sler and co-workers,26 DMSO is probably better than water in mimicking this environment. Moreover, the viscosity of cytoplasm (5-30 cP) is markedly higher than that of pure water (1 cP) .27 Then, the structures obtained in DMSO (7 = 2.2 cP) , if any, should be closer to the significant biological conformation than the structures in aqueous solution.

One of the first steps in the characterization of structural organization in the consensus domain would be to visualize a decrease in its mobility through the measurement ?f the correlation times ( 7 , ) , which should be greater in rigid regions. These r, values can be reached from the variations of the NOES with the temperature. Furthermore, this study allowed us to discuss the bias introduced in the in- terpretation of nmr data into molecular restraints by using an average correlation time all along the molecule.

MATERIAL A N D METHODS

Samples

Hexapeptide HERDVRGNH, and undecapeptide HERDVRGFASFLNH, were obtained by solid phase synthesis method.” They were then purified by high performance liquid chromatography and checked by several analytical methods.6

DMSO-d6 (99.97% D content) was used because of the insolubility of the undecapeptide in water.

UNDECAPEPTIDE REPRODUCING CONCENSUS SEQUENCE 142 1

The high viscosity of this solvent ( qDMS0/qwater = 2.2 at 25°C) led to an increase in the correlation times and allowed us to steer clear of the domain where NOES cancel out (i.e., 7, - 0.6 ns, at 300 MHz). Concentrations were around 10 m M for the undecapeptide and 15 m M for the hexapeptide. This second compound has also been studied in H,O and DzO.

The trifluoroacetic acid (TFA) content in the peptide samples was measured by a lgF-nmr method." Hexapeptide and undecapeptide contain respectively 2.3 and 2.5 molecules of TFA per molecule of peptide, suggesting that all the side chains were in an acidic form ( Arg, Asp, Glu) and that the N-terminal amine was more or less protonated.

Acquisition of NMR Data

Spectra were recorded on a Bruker AM-300 with a temperature control unit BVT-1000 and a 'H-l'F dual probe. Phased COSY diagrams have been obtained through the routine sequence with double quanta filtration (COSYPHDQ) . In water, presa- turation of the solvent was applied during the relaxation delay.

In the undecapeptide study, phased NOESY sequence has been modified following Rance et al.30 to reduce the scalar zero quantum (ZQ) artefacts, or J peaks, without increasing the tl noise. In these NOESYPHJ experiments, " the sequence was scaled to suppress the artefacts resulting from the JHNCH coupling constants ( J N J . Finally, NOESY diagrams have been obtained for a mixing time 7, of 400 ms and at 5 temperatures (293, 298, 308, 313, and 318 K ) .

Process of NMR Data

Data were processed on an As-X32 station through the UXNMR program. Free induction decays have been multiplied by shifted sine-bell functions and

Table I Example)

Process of NMR Data (Undecapeptide

2D Spectrum COSY NOESY

Dimension F1 F2 F1 F2 Window function sin2 sin2 Shift .rr/4 .rr/3 Acquisition size 512 2K 512 2K Transform size 2K 8K 4K 4K Digital resolution (Hz/pt) 2.8 0.7 1.3 1.3

digital resolutions improved by zero filling prior to Fourier transformation (Table I ) .

For NOESY experiments, the choice of a 7 ~ / 3 shift rather than the standard 7 ~ / 2 has already been discussed." Baseline corrections have been applied with a third-order polynom and finally the NOESY maps were symmetrized.

In water, chemical shifts are referred to 2,Z-di- methyl-2-silapentane-5-sulfonate sodium salt (DSS) , using a valine methyl resonance as inter- mediary reference in two-dimensional ( 2D ) experiments, while in DMSO, the DMSO-ds 'H peak was assigned to 2.52 ppm.

Molecular Modeling

Simulated annealing studies were performed using the standard conditions of the NMR Refine module in the Biosym package. Calculations were based upon the CVFF force field and typically force constants of 20 kcal/mol/A2 (or rad') for the experimental restraints. For the early stages of the process, i.e., until the final minimization, the nonbonded po- tential was reduced to a purely repulsive term ( quartic ) .

As the starting structure was a completely random array of atoms, a minimization phase preceded the dynamic run at 1000 K ( 15 ps) . In the first stage of molecular dynamics, the covalent and restraints constants were progressively scaled up, from low to full values, while the nonbonded repulsive term was kept very low. Then, in the second stage, the quartic constant was increased until its normal value.

Final minimizations were performed with full CVFF force field (Morse and Lennard-Jones po- tentials, coulombic term) , until a maximum deriv- ative of 0.001 kcal/A. Twenty independent structures were generated to test each of the hypothesis.

RESULTS

Chemical Shifts and 1 Coupling Constants

Assignments of the nmr Spectra and Chemical Shifts (Tables I / and I / / ) . The whole set of the spin sys- tems for the undecapeptide has been characterized by COSY ( a discrimination between Asp and Phe residues came out from the high frequency of the HPs, at more than 3 ppm, which could only be assigned to Phe residues). Finally, in the NOESY maps, sequential H a ( i) - - NH ( i + 1 ) , or aN correlations, allowed the specific assignments to Arg5

1422 BARON ET AL.

Table I1 Hexapeptide: NH, Ha, HO, and Hy Chemical Shifts (ppm)"

Residue Glu Arg5 ASP Val Arg8 GlY

Solvent H20 DMSO H 2 0 DMSO H20 DMSO H 2 0 DMSO H20 DMSO H20 DMSO

N H a 8.78 8.63 8.63 8.33 8.18 7.98 8.43 8.33 8.35 7.99 Ha! 4.10 3.85 4.35 4.36 4.72 4.63 4.14 4.10 4.34 4.12 3.92 3.64 HPI 2.17 1.94 1.80 1.51 2.79 2.43 2.09 2.07 1.80 1.62 HP2 Idem Idem Idem 1.65 2.87 2.69 Idem 1.69 HY 2.52 2.35 1.65 1.52 0.92 0.97 1.65 1.52

a Temperature: 303 K (water); 298 K (DMSO).

and Arg8, and to PhelO and Phel3 residues (Table 111).

The hexapeptide studies showed several problems. In water, 2D maps were very noisy, due to the amplitude of the residual H 2 0 peak. Moreover, the NOEs are weak at 18°C. The few observed NOEs were positive, i.e., 7, < 0.6 ns (the vanishing value for the NOE at 300 MHz) . It was necessary to heat to 30°C to detect univocally the sequential correlations GluHa - - ArgNH, leading to the Arg5 spin system, and ArgHa. - -GlyNH, then to the Arg8 one (Table 11). Finally, in DMSO, the overlaps of Val and Gly NH resonances (7.98-7.99 ppm), and of Arg8 and Asp NHs (8.33 ppm) , precluded further useful investigations of that sample.

The chemical shifts gave some information about the structural organization of these oligopeptides. For the hexapeptide in water, the chemical shifts of Arg5 and Arg8 were still degenerated (but for NHs because of the Glu NHi-Arg5 NH neighboring) while, in DMSO, differences were yet observed for H a and H/3 of the Arg residues. Moreover, in the undecapeptide, methyls of Val and H a of Gly were no longer chemically equivalent. These observations could indicate that the stabilization of some structure ( s ) increased in the order: hexapeptide (water)

< hexapeptide (DMSO) < undecapeptide (DMSO) . Finally, between the two peptides in DMSO, the chemical shift variations of interest were from the H a and NH of Val and Arg8, two residues in the center of the consensus domain.

NH Chemical Shift Temperature Coefficients, dBNH/ dT (Table IV) . Low absolute value of d S N H / d T , i.e., 5 5 ppm/K in water or 5 3 * lop3 ppm/K in DMS0,27,31 could indicate an involvement of the NH in an intramolecular H bond, at least in some com- formers whose weight could not be neglect.

The NH chemical shifts measured at 8 temperatures (between 293 and 318 K ) varied linearly and allowed the calculation of the d & H / d T values (Ta- ble IV). The hexapeptide, in water as well as in DMSO, did not show any stable H bond, while in the undecapeptide, Arg8 and Gly NHs were probably engaged in some interactions. This focuses once again on residues in the consensus sequence.

Finally, it was unnecessary to obtain other results about the hexapeptide because ( a ) previous information is in favor of random coiled features, ( b ) overlaps of the NHs would make elusive the analysis of most of the NOE data, and ( c ) this peptide is not a substrate for the endopeptidase. Nevertheless, its

Table I11 Undecapeptide (DMSO-d6, 298 K): Chemical Shifts (ppm)

Glu Arg5 Asp Val Arg8 GlY PhelO Ala Ser Phel3 Leu

8.67 8.41 7.83 3.87 4.38 4.62 4.17 1.95 1.56 2.46 2.05 Idem 1.68 2.72 2.38 1.52 0.81 Idem Idem 0.85

3.10 Idem 7.71

8.14 7.98 8.07 8.26 7.92 8.08 7.95 4.20 3.64 3.71 4.54 4.33 4.30 4.50 4.22 1.57 2.76 1.21 3.51 2.87 1.47 1.66 3.03 3.60 3.10 Idem 1.52 1.57 Idem 3.10 0.84 Idem 0.89 7.71

UNDECAPEPTIDE REPRODUCING CONCENSUS SEQUENCE 1423

Table IV Temperature Coefficients of NH Chemical Shifts (Unit: ppm/K; range: 293-318 K)

Arg5 Asp Val Arg8 Gly PhelO Ala Ser Phel3 Leu

Hexapeptide water 5.7 6.9 7.6 7.1 7.3

Undecapeptide DMSO 3.5 3.9 4.6 2.8 3.0 4.7 5.6 5.3 4.7 5.4 Hexapeptide DMSO 3.9 4.5 4.2 4.5 5.6

study gave a background of data that helps elucidate some features of the undecapeptide structure.

Scalar Coupling Constants, J (Table V) . From the fine analysis of cross peaks in the phased COSY diagram, most of the scalar constants have been determined and the more relevant Js in the undecapeptide are reported in Table V.

J N a depends on the Q, angle of the r e s i d ~ e . ~ ~ . ~ ~ Taking into account the two coupling constants for Gly, the best agreement is +70/80". In other residues, the large values of J N a leads to Q, = -120" (k40") ; in fact, probable CP values must be approx- imatively -150/140" or -100/90", except for Val ( CP - -120" ) . Other nmr data, i.e., some NOE correlations, depend also on Q, angles and a more ac- curate determination of these angles, taking into account all the data, will be presented in the discussion.

The small value of Jao in Val can be explain by a skew arrangement of Ha and HP, i.e., rotamer t or gt (Figure 1 ) . In Phe residues, Jupl (large) and JaB2 (small), lead to a predominant rotamer t or g- (Figure 1). Additional NOE data are necessary to specify these conformations.

NOE Interactions in the Undecapeptide

Normalized NO€ Data. Volumes of diagonal peaks, when possible, and of the more relevant NOE correlations, have been calculated, taking into account the overlaps in a practical way.25 Some correlations have been systematically measured, inside a residue, NHi- - - H a i ( N a ) , NHi. * .HPi (NO), and

HPi - - - HP'i ( P O ) , or between two adjacent residues, H a i . . . N H i + l ( o t N ) , H P i . . . N H J + l ( P N ) , a n d NHi * * * NHi + 1 (NN) . Other correlations involved the methyls of the valine residue (Ny and yN) and various interactions between nonadjacent residues, i.e., at medium or long range. For example, in a NOESY map obtained at one temperature, we measured 133 expe>rimental volumes of which 30 con- sisted in overlaps. Finally, we could calculate 30 diagonal peaks (out of a total of 40), 61 systematic NOE (out of 68 of interest) and 1 2 other correlations.

The NOE volumes have been normalized to the corresponding diagonal volumes, which permit US

to take into account most of the relaxation pro- and allowed a comparison of the experi-

ments at 5 different temperatures. In these conditions, a linear relationship between the normalized NOE (aAB) and the mixing times T , up to 500 ms, is generally ~ b s e r v e d ~ ' , ~ ~ , ~ ~ ; then, UAB / T, = GAB,

which is the cross-relaxation rate. GAB is proportional to the geometric factor 1 / r :B, 38939 rAB being the interproton distance.

In this analysis we could not quantify 10 of the diagonal peaks because they present severe overlaps. In these cases estimated values were obtained from data concerning the same chemical kind of proton in the adjacent residues. Moreover, 7 correlations could not be measured, because of strong overlaps (3 cases) or too small chemical shift differences (aa ,

ceSS 25,34-36

PO, "1.

NO€ (aAB) vs Temperature: Apparent Correlation Times 7,. Table VI shows some examples of variation of NOE with temperature. To rationalize them,

Table V Undecapeptide (DMSO-d6; 298 K): Selected J Coupling Constants (Hz)

Glu Arg5 Asp Val Arg8 Gly" PhelO Ala Ser Phel3 Leu

J(HNCaH) 7.9 8.6 10.4 8.9 5.8 5.6 7.8 7.3 7.9 7.8 8.2 J ( H a , HP,) 6.4 6.2 5.2 b b 9.5 6.9 5.9 8.9 7.4

b b 4.8 6.1 4.4 Idem J (Ha , HP,) Idem 7.3

'First entry, Hal (low frequency), second entry, H a 2 (high frequency) about 7 Hz 2 1 Hz.

1424 BARON ET AL.

Side-cha in of V a l i n e

g+ -

9 t

Side-cha in R = CH -C8

Rotamers around Ca-CP in the side chains

28

Figure 1. of amino acids.

an approximate relationship can be derived from the following:

1. The expression of the cross-relaxation rate ~ A B , under certain condition^^^,^^:

Q is a constant; I~zAB a multiplicity factor: in this study, 4/3 for NH/CH2 and 3/2 for NH/ CH3 interactions4'; w is the angular velocity of the radiofrequency; and TAB is an apparent correlation time of rotation for the vector r A B

in the magnetic field. The existence of a linear relationship between TAB and 7 / T, 39 where 7 is the viscosity of the DMSO (at the temperature T) : then 7 A B

= 72~*(7l /T) / (S" /T") , Or TAB = 72~'x, with X = f ( T ) = (v/T)/(o"/T"). The use of an approximation, a A B = CAB - 7,, 34

which was valid in related system^,^^,^^ but in some cases of pp correlations.

Then, the temperature dependency of most of the NOE interactions could follow the relation

X ( T ) were calculated from recently published values of 7 vs. T,39 with To = 293 K, the lowest temperature studied, and the data a A B = f ( x) analyzed with a least-square deviation process, using r iB as a fitting parameter. The 7& is generally longer, up to 2.9 ns (Figure 2 ) , than the mean correlation time, which could be estimated with the model of a spheric

Table VI Other Correlations of Val Residue)

Undecapeptide: Diagonal Normalized NOES" vs Temperature (Examples: (YN Correlationsb;

NOE Type 293 K 298 K 308 K 313 K 318 K

Hai. * *NHi + 1 Glu - - * Arg5 Arg5 - - - Asp Asp - - * Val Val * . . Arg8 Are8 * * Gly Gly (1) - . PhelO Gly (2) - * * PhelO PhelO - - - Ala Ser * - - Phel3 Phel3 - * * Leu

Val NH. * *Ha N H - - *HP N H - - - H y (1) NH - * * NH (Arg8) HP- * .NH (Arg8) Hy (1) .NH (Arg8) Hy (2) - - * NH (Arg8)

18.13 16.45 11.65 15.39 17.50 11.17 13.71 19.07 9.64 2.48

2.86 1.79 2.08 4.16 1.76 0.63 0.49

14.01 14.92 12.24 14.62 15.84 9.69 6.23

12.13 6.41 2.02

3.61 1.56 1.51 5.53 1.35 0.67 0.74

6.35 10.49 9.00 9.23

11.35 6.70 4.77 7.64 3.36 0.14

2.00 2.21 1.52 4.46 1.53 0.12 0.26

2.85 7.65 7.02 9.77 9.29 4.91 3.53 5.54 1.76

-0.45

2.44 0.39 0.30 3.35 0.48

-0.02 0.08

0.94 6.66 6.47 7.48 7.31 4.35 2.29 4.18 0.80

-0.57

1.46 0.25 0.12 1.41 0.35

-0.02 -0.18

a CZAB in '36 of the mean diagonal peak. But Ala * * Ser (overlap with Ser N a ) .


00 0 0 0

Figure 2. Undecapeptide (DMSO-4). Apparent correlation times, 7 i B (293 K) , from the fits NOEs vs temperature. NOEs ( uAB) have been normalized against the mean diagonal peak. T, = 400 ms. UAB = ~ [ 6 / ( 1 + 4 w 2 . ~ ~ ~ . x X 2 ) - 1 ] . X , w i t h X ( T ) = f ( T ) = ( q / T ) / ( q O / T o ) and To = 293 K.

and rigid molecule, 7, = ( 4 7 r R 3 / 3 ) q / ( k T ) , i.e., 0.8 ns for this undecapeptide in DMSO at 293 K.

Figure 2 shows the major trends for the variations of T&, which reflect the degrees of freedom in the molecule. The maxima are found in the Asp-Val- Arg8 segment and the minima in the N- and C-ter-

minal parts. The dissymmetry of the diagram suggests a stabilization of the structure in the consensus domain rather than the result of statistical combined motions. Otherwise, when a side-chain proton is involved in a correlation (NP, PP, PN, Ny, y N ) , the r iB value is generally smaller, as expected, than those of the backbone correlations (Na , NN, a N ) in the same residue.

Interproton Distance Factor (Q/ r '): Model with Several 7,. In order to soften the effects of the fitting parameter T&, on the determination of & / T i B , the geometric means of T& were calculated for each residue and for two classes of correlations: ( a ) between backbone protons only and ( b ) HP involving interactions (Table VII). Hi. - .NHi + 1 correlations (NN, a N , and ON), which depend on the P angle of the ith residue, have been pooled with the corresponding interactions in the residue i. Some values have been discarded from these means, when the fitting parameter r.& was too large to have any physical meaning or when the accuracy of the fit was too bad to allow the determination of the geometric factor with some confidence level (Table VII), for example, some correlations of the C-terminal fragment, Ser-Phel3-Leu.

The averaging procedure gave a better visualiza- tion of the previous discussed trends, even if some details could be rubbed out. Moreover, keeping rZB at the corresponding value in Table VII, a new analysis of (.IAB = f ( X ) leads to geometric factors ( Q / r iB) whose internal consistency would be better than those obtained from the first calculations; this permitted us also to analyze, in a better way, the data of two correlations (NN Asp - - - Val and Ncu Arg8) whose former T,& values were meaningless.

Finally, an accuracy of the interproton distance factor ( Q / r 6 ) could be estimated from a search of a limit of this factor, which brought about the upper

Table VII Undecapeptide (DMDO-d6; 293 K): Correlation Times T:~ (ns); Mean Valuesa

Glu Arg5 Asp Val Arg8 Gly PhelO Ala Ser Phel3 Leu

Backbone ( N a , NN, aN) 1.0 1.2 2.1b 2.4 1.6" 1.4 1.2 1.1 1.1 0.9 0.9 Number of NOEsd 1 3 2 3 2 4 2 2 1 1 1

Side chain (NP, PP, PN) 0.9 1.0 1.1 1.2e 1.1 1.1 1.0 0.9 0.8 Number of NOEsd 1 4 5 2 4 5 2 3 3

a Geometric means. I, NH (Asp) * * 1 NH (Val) discarded. ' N a discarded.

Taken into account in the mean. N y and yN: 1.0 ns (3 values).

1426 BARON ET AL.

Table VIII Several Correlation Times (NAB) or with the Same Correlation Time (NhB)a

Undecapeptide (DMDO-d6): Normalized NOES (% of 1.75 A Ha * . H Interaction) Model with

Model Several Correlation Times Same Correlation Time

NOE Type Correlation NAB ANAB d- d+ N X B ANXs d- df

NP"

NTd NN"

aNf

Nab Arg5 ASP Val Arg8 Gly (1) G b (2) PhelO Ala Phe l3 Leu Arg5 (1) Arg5 (2) ASP (1) Asp (2) Val Arg8 (1) Arg8 (2) PhelO (1) PhelO (2) Ala Ser (1) Ser (2) Phe l3 (1) Phel3 (2) Val (1) Arg5 - - - Asp Asp - * * Val Val - - * Arg8 Arg8 - * - Gly Ala * - - Ser Glu . - - Arg5 Arg5 * - Asp Asp - - - Val Val * - - Arg8 Arg8 * - Gly Gly (1) - * -PhelO Gly (2) * - - PhelO PhelO- - -Ala Ser - * - Phel3 Phe l3 - - Leu

4.6 3.2 2.6 6.3

12.3 10.5 7.2 7.7 6.0 4.4

15.4 3.6

14.3 10.3 3.7

15.3 14.7 18.1 5.0

10.6 7.0 7.5 7.5 2.0 5.5 2.2 1.9 3.9 3.8 6.2

45.7 37.4 11.6 11.8 21.7 17.0 15.3 33.4 21.4 9.4

22 4 9

11 4 9 4 3

31 23 15 32 6

13 24 5

10 10 14 3 8

10 10 29 15 23 24 12 3

13 2

11 2 1

1 16 6 4

10

limit of the sum of square deviations, S = So (1 + f ) , where So was its minimum and f represents the reverse of the degree of freedom in these fits with 5 data and two parameter^.^^,^^ For 54 correlations, the uncertainty on the geometric factor was <32%, with an overall mean of 12%.

correlations (Asp, PhelO, Phel3), a Q / r 6 mean was calculated and used to normalize all the results as N A B , percentages

From the three available

2.8 3.1 3.2 2.7 2.5 2.5 2.7 2.7 2.7 2.8 2.3 2.9 2.4 2.5 2.9 2.4 2.4 2.3 2.8 2.5 2.7 2.6 2.1 3.2 2.8 3.2 3.3 2.9 3.0 2.7 2.0 2.0 2.5 2.5 2.3 2.3 2.3 2.1 2.2 2.6

2.6 2.7 2.9 2.9

2.5 3.9 2.5 2.8 3.7 2.5 2.5 2.4 3.3 2.7 3.0 2.9 2.9

3.2 4.3 4.5 3.5 3.5 3.1 2.0 2.1 2.6 2.6 2.3 2.4 2.5 2.1 2.3 2.8

6.3 9.3 8.1

11.6 20.2 16.5 8.8 8.8 3.9 2.8

13.6 3.4

15.5 10.4 4.8

16.9 16.2 17.1 5.2 9.1 4.5 4.8 4.2 1.0 5.0 2.8 5.0

12.9 7.7 6.8

42.8 40.4 30.2 37.8 42.9 26.8 26.8 41.3 21.4 6.0

11 5

12 8 7

11 8 6

33 27 15 25 9

15 27 8

12 6

16 6

11 13 12 21 17 25 23 16 6

16 5 3 5 4 1 3

18 10 8

12

2.7 2.6 2.6 2.5 2.3 2.3 2.6 2.6 2.9 3.0 2.4 3.0 2.4 2.5 2.8 2.3 2.3 2.3 2.8 2.6 2.9 2.8 2.9 3.7 2.8 3.1 2.8 2.4 2.7 2.7 2.0 2.0 2.1 2.0 2.0 2.2 2.1 2.0 2.2 2.7

2.8 2.4 2.5

2.7

2.6 3.0

2.5 2.6 2.5 4.1 3.0

4.2

4.2

4.4 2.8 3.2 3.6 2.0 2.0 2.2 2.1 2.0 2.2 2.3 2.1 2.4

of the NOE relative to a pair of protons distant from 1.75 A (Table VIII).

lnterproton Distance Factor (Q/r '): Rough Model with the Same 7,. Most of the NOE studies are done at only one temperature and analyzed with the hypothesis of an overall 7,, the same for all the pairs of protons. The present data can be used to evaluate the uncertainty brought about by this rough model.


Table VIII (Continued)

Model Several Correlation Times Same Correlation Time

NOE Type Correlation NAB ANAB d- d' N)AB A ~ A B d- d+

PNg Glu - - + Arg5 Arg5 (1). * .Asp Arg5 (2) - Asp Asp (1)- - .Val Asp (2) - * . Val Val. . - Arg8 Arg8 (1) - .Gly Arg8 (2) - - * Gly PhelO (1). - -Ala PhelO (2). * .Ala Ala * * Ser Ser (2) * - - Phel3 Val (1) * - eArg8 Val (2) - - - Arg8

YNh

3.6 6.0 3.9 3.6 3.5 3.2 5.1 5.9 3.8 7.0 4.7 7.3 1.7 1.7

13 18 5 5

13 16 7 2

15 8 8

22 16 21

3.0 3.6 2.7 3.2 3.0 3.5 3.0 3.5 3.0 3.7 3.0 3.8 2.8 3.2 2.8 3.1 2.9 3.6 2.7 3.0 2.9 3.3 2.6 3.1 3.4 4.6 3.3 4.7

2.3 5.0 3.4 4.0 3.7 4.4 5.9 6.7 4.1 7.2 4.2 4.7 1.7 1.6

17 20 8 5

16 19 9 6

11 10 9

25 16 23

3.2 2.8 4.3 3.0 3.0 4.4 3.0 2.9 4.5 2.8 3.7 2.7 3.4 2.9 4.5 2.7 3.4 2.9 4.4 2.8 4.6 3.4 3.4

a Accuracies ( A N in % of N ) and distance bounds (in A): d-, from the rigid model, and d+, from the fluctuating models (rapid N, slow: N'). Some unrealistic d+ have been discarded.

But Ser (overlap). 'But Leu (too weak).

Val only, Ny(2) too weak. But Gly. . -PhelO (too small chemical shift difference), and PhelO- * .Ala, Sera * aPhel3, Phel3. * .Leu (too weak). But Ala - . * Ser (overlap). But Ser (1). . . Phe 13 and Phe 13 (1 and 2 ) . . .Leu (too weak) Val only.

When the rough NOEs are negative, experimental data at the lowest temperature are the more appro- priate to be considered, in order to deal with the largest absolute NOE values (Table VI) . However, to soften the experimental uncertainties of a single measurement (a t 293 K ) , the theoretical computed value from the first a A B = f ( X ) analysis was used (i.e., using the r iB values of Figure 2 ) . Finally, the ratios with the mean of the three pp correlations (293 K data) are reported in Table VIII as N L B .

The comparison of the two sets of normalized NOEs in Table VIII shows that the biggest discrepancies are observed at the level of the backbone correlations in the consensus domain, with ratios N L B / N A B always being 21.6 from Asp to Gly, and a maximum of 3.1 to 3.3 for the Val residue. These problems are quite directly correlated with the 7;B:

Knowing that for the mean of the pp correlations, ripp correspond to about 1.1 ns, when 0.9 2 T ~ B 2 1.2 ns the differences between N L B and N A B remain small, i.e., for most of the HP involving correlations and some backbone interactions. But when 1.4 2 rZB 2 2.4 ns, neglecting the variation of the frequency factor ratio ( 17% ) , the discrepancies are quite directly proportional to r i B :

,

DISCUSSION

Side-Chain Rotamers

Generally, NOE NO are needed along with JaB to assign the right predominant rotamer about Ca-Cp. As illustrated in Table VIII, NOE NO values were not too sensitive to the model used for the normalization and at least relative values of NO1 and NP2 could be enough for the discussion. In the Val residue, where Jas leads to rotamer t or g', the small NO value shows a trans arrangement for NH/HP.

1428 BARON ET AL.

This suggests the existence of a single rotamer, gt (Figure 1 ) . For the Phe residues in which rotamer g' was eliminated from Jmp values, the big difference between NP1 and NP2 leads to the predominance of the g- conformer and to stereospecific assignments, i.e., HOD is HP1 and HPL is HP2 (Figure 1 ) . Finally, it is also possible to suggest the predominance of the conformer gi in Arg8 because of the two large NP correlations, while their clear difference in Arg5 would indicate t or g- ( Jap are not available and this does not allow us to discriminate between the two possibilities).

Relationships Between NOEs and lnterproton Distances

The peptide conformation depends on the @ and \k angles of the residues and these angles are related to the arrangement around sp2-sp3 atoms, which cannot be reduced, even in a first step, to the choice between 3 staggered rotamers, as for the Ca-CP problem. Consequently, a qualitative relation between data and conformers is not yet sufficient and the NOEs must be translated into distances.

In fact, at least three models can be used for the relationship between the NOEs and the interproton distances; they depend on the flexibility of the molecule under consideration. Along with the limited model of a rigid molecule, where CTAB = ( - r i g ,43 two other models take into account the variation of rAB. If an equirepartition of the distances rAB is postu- lated, between the limits rmin and rmax, 25,43,44 the following relationships can be derived

1. for slow fluctuations, without influence on the 7, Of each rAB,

2. for very rapid fluctuations, leading to a decrease of the corresponding r,,

It is obvious, from the results in Figure 2, that the rigid model is inadequate in this oligopeptide, but it could serve to define, for a given NOE, the shortest value of the interproton distance. As in this study it is possible to take into account the motions, a realistic uppest value for rAB could be calculated from the model based on rapid motions ( N A B values only).

Finally, in Table VIII, the accuracies of the de- terminations of N A B (and NLB) are used to calculate,

from ( N + AN) and the rigid model, the lowest bound for the interproton distance d - , and from ( N - AN), its upper limit d + , using either the rapid motion model for NAB or the more general slow fluctuation relationship for N L B . In those conditions, only real constraints would be set down in the view of the molecular modeling phase.

Peptide Backbone Conformational Analysis: and \k Angles

In each residue, the coupling constant J N n and the NOEs Na, NP, and N N are correlated to @, while the NOEs NN, PN, and a N depend on \k. Then, the best set of @ and \k angles can be obtained by minimization of a"pena1ty function, Z A d 2 , where Ad is the difference between the theoretical interprQton distance for a given value of @ or \k or both (the case of the NN interaction) and a limit in Table VIII, if the theoretical distance is outside the re- straint domain."

One problem is the translation of the J ~ J ~ into restraints of same weight as NOE data. The simplest way, from J N a to @ and then to dNa is not a satis- factory one because between -150" and -80" dNa is staying around 2.9 A, while J N a varies from 7.9 to 9.7 Hz, markedly more than the uncertainty on the coupling constant, about k0.5 Hz. Finally, we chosed to convert the A J (difference between the experimental and the theoretical J for a given value of @) into a pseudodistance violation Ad with the scaling factor 0.1 w/0.5 Hz.

The best sets of @-\k, in Table IX, show that the @ values are not too sensitive to the normalization model used, which reflects the weight of J N m data in the search, with @close to -150/140" or -100/90". On the other hand, \k depends on these models, especially in the Asp-Val-Arg8 fragment, where \k(NLB) is less than * ( N A B ) for at least 20", and up to 60" in the Asp residue. In the latter case, the lessening of the a N correlation ( N A B compared to NLB) leads to 2.5-2.6 b, for dmN (2.1-2.2 b, from NLB), and ruled out the +110/130" domain. Oth- erwise, in the C-terminal part of this peptide, where the NOES are generally smaller, the slow fluctuation model brings about greater uncertainties on the distances and so on the Q values.

Structural Elements of the Consensus Domain

Five kinds of results tend to prove a structural organization of the 5-9 fragment in the undecapeptide, even only in minor conformers:


Table IX Undecapeptide (DMSO-D,): Optimal Sets of and * Values, from JNn and NOES

Model NOEs Sets

Several Correlations Times, NAB

Same Correlation Time, N X B

9 @ 9

Glu Arg5 ASP

Val Arg8 GlY

PhelO

Ala

Ser

Phel3

Leu

-140 - 140/120 -100 -90

-130 -150

-80/70 +70/80 -90

-80

-150 -90 - 150/90 -150/90 -150/140 -100/90

+ 120/130 $130 +170 + 170/180 $170 $80

+120/130 $170 $170 f140

-60

+ 140 f140 -180/170 +40/60

-150/90 -140 -100 -90

-140 -150 -80/70 +70/80 -90 -90 -80 f 8 0

-150 -90

-150

-150/140 -100/90

+110/130 +110/130 +110/130 +110 +110

+60 + 100 +170 +170 +130 f10/60 -60 0/+50

+90/160 +80/160

-180/+50

1.

2.

3.

4.

5.

Chemical shifts are different for the methyls of Val and the Ha of Gly, and we observed variations of chemical shifts between hexa- and undecapeptide, for Ha and NH of both Val and Arg8; NH chemical shift temperature coefficients are significantly brought down for Arg8 and GlY; 7, value of backbone interproton vectors are clearly higher in the Asp/Gly segment ( 1.4- 2.4 ns), and especially for Asp-Val (2.1 and 2.4 ns) , than in other residues (0.9-1.2 ns) whose values are close to those involving side chain protons (0.8-1.2 ns) ; \k value of Val indicates a folding tendency, even in the average structure; NOEs between nonadjacent residues, Glu - - Gly and Asp * - - Arg8, are observed through the studied temperature range (Ta- ble x); even small ( N A B ) and very labile ( T & ) , these interactions are good qualitative proofs of the folding possibility of the consensus sequence.

interactions would be expected with this 0-turn, especially Asp Ha * * - Arg8 NH (found only at 293 K, with a 1.5% level) and a strong NN between Val and Arg8 (expected distance 2.4 A ) , which seems excluded from the present data but could be accepted by removing the 293 K point in the analysis of this correlation (Table VI) . Otherwise, one can wonder about the effect on the 0-turn like structure of the second H bond, between Gfy NH and Glu CO. AC- tually, molecular modeling using a simulated annealing protocol derived from Nilges et al.45 showed that the energy of the sheet with two H bonds was about 40 kcal higher than those of both the 8 --* 5 0-turn and the 9 --* 4 loop.

In order to get more information about these folded structures, other molecular modeling studies

Table X Undecapeptide (DMSO-d6): NOEs Between Nonadjacent Residues*

TAB

(ns, 293 K) NAB

Glua - * * Glya (1) 0.34 1.6 0.29 1.5 Glua. . . Glya (2)

tionalized by an extended @-turn, from Arg5/Arg8 AspNH. . , Arg8NH 0.59 2.5

In a first stage, most of these data could be ra-

to Glu/Gly with H bonds between Arg8 NH - - - OC -

Arg5 and Gly NH * - - OC Glu (Figure 3 ) . But other a Normalized against 1.75 A H . . . H NOE.

1430 BARON ET AL.

-NH-Co< -C-NH-Co( H-C-NP-CCX H II : I 0 : co II

0 I

H .. I .. H *. ‘fil I ’. ‘I’

I NH

I -CO-Co< ?l-N-CO-Co( H-N-CO-Cd H

( A r g 8 1 ( V a l 1 Figure 3. Undecapeptide (DMSO-4). Schematic structure of the consensus domain with a putative 5-8 p- turn and an additional 9 + 4 H bond. NH with low temperature coefficient of the chemical shift are circled. Me- dium range NOES are indicated with dots.

would be necessary: ( a ) starting with the’ @-!P sets of Table IX and using molecular dynamics under restraints to generate actual conformers; ( b ) dis- criminating the conformations upon the H-bonding availability of the NH of Arg8 and Gly, the neighboring of Glua and Glya, etc.; and then (c) trying to analyze the nmr results as a combination of two conformers, as already outlined by Amodeo et al.46

So far, the present results revealed the propensity of this synthetic peptide to organize structures in the DMSO solution and suggests an equilibrium between two folded conformations stabilized by one H bond in the consensus domain. The relationship between the residues implied in the conformational organization and those required at the cleavage site of the RXVRG endoprotease leads us to hypothesis that the conformations drawn from the nmr study allow the precursors to be most adequately fit for endoproteolytic cleavage.

We thank Drs. C. Creminon and H. Boussetta for the synthesis of the peptides, Dr. J. Vickrey for helpful com- ments on the manuscript, and we are grateful to Prof. P. Cohen for his continuous interest in and helpful advice for this work.

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95-117.

Received October I , 1993 Accepted April 14, 1994

Conformational studies of an undecapeptide reproducing the consensus sequence around the cleavage...

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