'H NMR-based Determination of the Three-dimensional Structure of ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Val. 268, No. 12, Issue of April 25, pp. 8580-8589.1393 Printed in U.S. A.

0 1993 by The American Society for Biochemistry and Molecular Biology, h e .

‘H NMR-based Determination of the Three-dimensional Structure of the Human Plasma Fibronectin Fragment Containing Inter-chain Disulfide Bonds* (Received for publication, December 1, 1992)

Leela Kar$, Ching-SanLaign, Carl E. Wolffg, David Nettesheimll, Simon Sherman$, and Michael E. Johnson$li From the $Center for Pharmaceutical Biotechnologyand Department of Medicinal Chemistry and Pharmacognosy, P. 0. Box 6998, University of Illinois, Chicago, Illinois 60680, the §Department of Radiology, BiophysicsSection, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and the 11 Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin53201

Human plasma fibronectin is a plasma glycoprotein adhesion and spreading, wound healing, phagocytosis, and that plays an important role in many biological proc- differentiation(Hynes, 1990; Mosher, 1989;Akiyama and esses. It consists of two identical 230-250-kDa sub- Yamada, 1987). The protein consists of two nearly identical units thatare joined by two disulfide bonds near their subunits of 230-250 kDa each, containing binding domains carboxyl termini.Each subunit contains variousbind- for various biomolecules including fibrin, heparin, collagen, ing domains composed of three types of homologous DNA, and cell surface molecules. The various binding dorepeats. Recent work has determined the three-dimen-mains of Fn are composed of homologous repeats of three sional structures of various repeat fragments, but little different types, namely, type I, 11, and 111, containing 45, 60, is known about the three-dimensionalstructure of the and 90 amino acids, respectively (Petersen et al., 1983; Korncarboxyl-terminal region. A recent NMR study of a blihtt et al., 1985). These three types of homologous repeats plasmin-digested carboxyl-terminal inter-chain disulfide-linked heptapeptide dimer has proposed that the are also found in other proteins involved in blood coagulation two subunits are arranged in an antiparallel fashion and in proteins of the extracellular matrix (Patthy, 1990). 2D (An et al. (1992) Biochemistry 31, 9927-9933). We NMR investigation has shown that theseventh type I repeat have now determined the three-dimensionalstructure of Fn (48 residues) primarily consists of two antiparallel pfor a substantial portion of a trypsin-digested inter- sheets (Baronet al., 1990). The type I1 repeats of Fn resemble chain disulfide-linked 52-residue (6 kDa) fragmentof the Kringle structure derived from the crystal structureof the the carboxyl-terminal of human plasma fibronectin prothrombin Kringle 1unit (Holland et al., 1987; Constantine (which includes the above-mentioned heptapeptide di- et al., 1992).The solution structure of the tenthtype I11 repeat mer) using two-dimensional NMR methods and a new (94 residues) has been determined by 2D and 3D NMR (Baron strategy for NMR-based protein structure determina- et al., 1992), and thecrystal structureof a Fn type I11 domain tion. The NMR data requires that the two chains in from the tenascin, an extracellular matrix protein hasbeen deterdimer be linked in a symmetric, antiparallel arrange- mined by x-ray crystallography (Leahy et al., 1992); both ment. The resultingmonomer conformation consists of structures consist of two antiparallel @-sheetswith an immutwo twisted or coiled segments, ThrS-Asns and Ilee- noglobulin-like fold. Phela, connected by the Cys7-Prosresidues in extended The two subunits of Fnare joined near their carboxyl conformations, with the two monomer chains cross- termini by two disulfide bonds. Despite recent progress in linked at residues Cys’ and Cys”. The conformation of obtaining detailed information concerning the primary setheheptapeptidedimer region differssubstantially quence and gene structure of Fn, and the three-dimensional from theconformations proposed by An et al. structures of the various repeat fragments, comparatively little is known about the three-dimensional structure of the carboxyl-terminal region. In particular, the spatial arrangement of the subunits about thesedisulfide bonds, i.e. whether Human plasma fibronectin (Fn)’ is a large glycoprotein the monomeric chains are arranged in a parallel or an antipresent in blood plasma at about 0.3 mg/ml. It plays an parallel fashion in the dimer, has not been conclusivelydeterimportant role in many biological phenomena including cell mined. A recent report, based on analysis of2D NMR data * This research was supported in part by grants HL45977, GM- for a 14-residue fragment of the carboxyl-terminal region, has 35719, and RR-01008 from the National Institutes of Health. The suggested that thetwo subunits arearranged in an antiparallel costs of publication of this article were defrayed in part by the fashion, and has proposed two alternative three-dimensional payment of page charges. This articlemust therefore be hereby structures for aqueous and dimethyl sulfoxide environments. marked “advertisement” in accordance with 18 U.S.C. Section 1734 There are, however, problems with the analysis and resulting solely to indicate this fact. 11 Current address: Abbott Laboratories, Dept. 47G,Bldg. AP9, structures presented in that work that are discussed in more detail in the discussion section below. Abbott Park, IL 60064. In thiswork, we have purified the carboxyl-terminal 6-kDa 7 To whom correspondence should be addressed. The abbreviations used are: Fn, fibronectin; 2D, two-dimensional; Fn fragment containing two 26-residue fragments with interNMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; chain disulfide bonds, and have determined the three-dimenCOSY, two-dimensional correlated spectroscopy; NOESY, two-di- sional structure of the disulfide linked region by2D NMR mensional nuclear Overhauser spectroscopy; d a ~ ( ji), , dNN(i,j ) , etc., intramolecular distance between protons C”H and NH, NH and NH, methods. We report NMR assignments for 25 of the 26 etc. on residues i and j; armsd, angular root mean square deviation; residues in the monomer of the 6-kDa carboxyl-terminal fragment. The NOE data and a new strategy for NMR-based HPLC, high-performance liquid chromatography. 8580

NMR-based 3 - 0 Structure of Fibronectin C-terminal Fragment protein structure determination have been used to build the three-dimensional structure of the Thr3-Pro14 segment containing the interchain disulfide bonds. We show that the NMR data are consistent with only one set of structures: those in which the two interchain disulfide bonds linking the monomers of the Fn molecule are arranged in an antiparallel fashion. The resulting structure for the disulfide-linked region of our 52-residue fragment differs substantially from those reported by An et al. (1992) for their 14-residue fragment. We suggest that thehighly truncated heptapeptide dimermay not retain its native conformation in either aqueous or dimethyl sulfoxide solution. EXPERIMENTALPROCEDURES

Sample Preparation-Fn was purified from fresh-frozen human plasma, obtained from the Blood Center of Southeastern Wisconsin, ona Sepharose 4B column and a gelatin-Sepharose 4B column, arranged in tandem (Engvall and Ruoslahti, 1977). The integrity and purity of the protein were routinely examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purification of the carboxyl-terminal 6-kDa fragment was performed essentially as described by Garcia-Pardo et al. (1984) with some modifications. Fn (typically 60 mg) was incubated with trypsin at a 33001 (w/w) ratio of Fn:trypsin for 30 min. Digestion was stopped by adding phenylmethylsulfonyl fluoride to a final concentration of 0.1 mM and soybean trypsin inhibitor at a 2:l ratio of inhibitor:trypsin. The material was then applied to thefollowing columns (2.5 X 8 cm, approximately 40 ml, flow rate of 50 ml/h) arranged in tandem: gelatin-Sepharose, heparin-Sepharose, and DE52. The final column was then washed with 10 mM Tris (pH 7.4), and 75 mM NaCl. The 6-kDa fragment, along with the 34-kDa fragment, was eluted with 10 mM Tris at pH 7.4, containing 0.5 M NaCl. The fractions werepooled, dialyzed, concentrated by partial lyophilization, and loaded onto a Sephadex G-50 column (1.5 X 95 cm, approximately 170 ml, flow rate of 8 ml/ h) for final purification. The 6-kDa fragment from this stepwas free of higher molecular weight fragments as judged by SDS-PAGE (silverstained, 15% acrylamide gel). The typical yield was about 0.5 mg of the 6-kDa fragment per 60 mg of plasma Fn. The amino acid composition and sequence of the 6-kDa fragment were determined and found to be identical with the 26 carboxyl-terminal residues of plasma Fn (Peterson et al., 1983). For NMR measurements, about 3 mgof the 6-kDa fragment was lyophilized to dryness and redissolved in 0.5 ml of either D20, or HzO containing 5% D20,giving a final peptide concentration of about 1 mM. 'H NMR Spectroscopy-NMR data were accumulated on a General Electric GN500 spectrometer equipped with a 1280 Nicolet computer. Phase-sensitive two-dimensional (2D) COSY and NOESY data sets were collected in the hypercomplex mode (States et al., 19821, with standard pulse sequences and phase expressions (Jeener et al., 1979, Wider et al., 1984). NOESY data were acquired with mixing times of 125 and 250 ms. Relayed COSY experiments in the absolute value mode (Wagner, 1983) were used to help identify spin systems of side chains. All 2D data sets consisted of 1024 complex points in the tS dimension and either 256 or 320 complex points in the tl dimension. All data processing was done on a Silicon Graphics IRIS computer, using the FTNMR software (Hare Inc., Woodinville, WA). Digital filtering was used prior to Fourier transformation in every case. All NOESY spectra were base-line-corrected by a fifth-order polynomial. Chemical shifts are referenced to thewater signal, which is 4.79 ppm a t 25 "C. from 4,4'-dimethy1-4-silapentane-I-sulfonate Computational Methods-Two different approaches were examined for the determination of spatial structure from NMR data: (i) the deterministicdistance geometry (DG) approach (Wuthrich, 1989), followed byenergy refinement; and (ii) a build-up strategy (BUILD), using a probabilistic model of protein conformation (Sherman et al., 1987,1988; Sherman and Johnson, 1993). The DSPACE software package (Hare Research, Inc., Woodinville, WA) was used in the DG approach, to generate structures consistent with covalency constraints and semiquantitative estimates of interprotondistanceconstraints, starting with randominitialatomic coordinates (Nerdal et al., 1988). 2.0 A was used as a lower limit of distance constraints, and 2.6, 3.3, and 4.0 A were used as upper limits for qualitatively observed strong, medium, and weak cross-peak intensities, based on the estimation of distances from the experimentally observed volume integrals for a sampling of NOE cross-peaks (using a proportionality constant derived from the volume integrals

8581

of the cross-peaks relating 6H and CH of PheL2across a distance of 2.5 A). Structure refinement was obtained by randomization of COordinates followed by a cycle of simulated annealing and conjugate gradient minimization of penalty functions. All atoms in the segment considered were subjected to simulated annealing. Similarly, all atoms were considered in the calculations of penalty function and gradient. Since the NMR data indicate a symmetrical structure, symmetry was used as a constraint in the energy refinement process. Additional structures were generated by random embeds, and theenergy refinement and simulated annealing algorithms were repeated to minimize the constraint violations for the new structures. A set of structures, generated by DSPACE, and selected qualitatively for their conformational diversity by comparison of ($,#) plots, was also used as starting structures for restrained energy minimization (DGREM) and restrained molecular dynamics (DGRMD) calculations using the CHAR" software package, with empirical energy potentials taken from Brooks et al. (1983). All calculations were performed on a Silicon Graphics work station. The BUILD approach uses, in addition to NMR data, a priori information on empirical distribution functions for backbone conformations, generated from the high resolution x-ray structures in the Protein Data Bank. NMR information regarding the presence or absence of sequential d connectivities (NOE cross-peaks among nearest neighbor residues) is used for statistical prediction of backbone conformations of individual amino acid residues. A three-step procedure was followed (i) estimation of a starting set of angular coordinates (local conformations) from the NMR data (Sherman et al., 1987); (ii) determination of the spatial structure by a gradual buildup process (Sherman et al., 1988); (iii) structurerefinement by energy minimization on unconstrained structures. The FISINOE program (Sherman and Johnson, 1992) was used to estimate the @,$values , with corresponding probabilities for each residue, given the d a ~dNN, and d # connectivities. ~ The most probable $,# values for each residue were used as the starting set of angular coordinates. The BUILD strategy to obtain a spatial structure from the starting set of backbone conformations utilizes an optimality principle in which the fragment considered a t any stage has a minimum number of residues and a maximum number of restrictions. Long-range NOE requirements and the value of conformational energy were used as steering parameters to guide the BUILD process. Two general assumptions were used: (i) t,he upper limit of distanceconstraints is 3.3 A for sequential d connectivities, and 4 A for long-range NOEs; and (ii) all dihedral angles must lie within sterically allowed regions in conformational space. For the sake of convenience, all NOEs other than sequential are termed long-range. The interactive graphics package INSIGHT (Biosym Technologies, San Diego,CA)was used to construct the starting structures, with backbone conformations estimated using FISINOE. All energy minimizations were performed using CHAR" on a Silicon Graphics work station. The force constant for the dihedral constraints was reduced in gradual steps from 50.0 (kcal. mol".rad-*) to 2.5 and finally to 0.0, decreasing it by about a factor of two following each cycle of 250 steps of conjugate gradient minimization. A macroscopic dielectric constant of 10 was used for these calculations. Calculations using dielectric constants of 1, 2, 4, 10, 30, and 80 showed that conclusions based on energy considerations were unaffected for dielectric constants greater than 4. Although the same starting values were used for the dihedral angles of a particularresidue in the two chains, symmetry was not used explicitly as a constraint in the energy minimization process. In general, distance constraints also were not included explicitly in the calculations, except where specifically stated. RESULTS ANDDISCUSSION

NMR Assignments-The

6-kDa carboxyl-terminal fragment from human plasma fibronectin has the following sequence for the 26-residue monomer, with two monomers joined either parallel or antiparallel to form the dimer Thr-

Asn-Thr3-Asn-Val-Asn-Cys7-Pro-Ile-Glu-Cys'1-Phe-MetPr~'~-Leu-Asp-Val-Gln-Ala-Asp-Arg-Glu-Asp-Ser-ArgAspz6. The Thr3 to Arg5 segment was readily assigned by a combination of2D NOESY, COSY, and RELAY COSY experiments in D20 andHzO. Resonances for G1uZ6and the amide resonances for Thr' and Asn'were not readily observable. The threonine and valine side chains were identified by a relay experiment in DzO that showed the cross-peak between

NMR-based 3-0Structure of Fibronectin C-terminal Fragment

8582

the C,-proton and the y-methyl protons, and also by their characteristicspin patterns and NOEs in the COSY and NOESY spectra, respectively. The asparagine side chains were identified by their chemical shifts in the COSY, and, in the case of Asn' and Am6,by NOEs between the 0-methylene protons and they-NH2group (Fig. 1).The two prolines (Pro' and Pro14) were identified by their characteristic cross-peak patterns in both COSY and NOESY spectra (illustrated for Pro' in Fig. 2). The lone isoleucine (Ile') was readily identified because of its unique spin system (Fig. 2). Similarly, Phe12 was easily assigned, being the only aromatic residue in the fragment. The ortho, meta, and para protons of Phe" were traced out in a COSY spectrum, and the@-methyleneprotons were identified by their strong NOEs to thearomatic protons (Figs. 1 and 3). Most of the peptide backbone of the Thr3Arg5 segment could be traced by &N connectivities in a 250-

r

.I

I

I

I

4.0

3.0

2.0

1.0

PPm FIG.2. Combined COSY-NOESY spectra showing the aliphatic region. Spectra were obtained in DzO at pH 7.0 and 25 "C, with a 250-ms mixing time for the NOESY experiment. The spin systems for Pro' and Ile9 are traced out using large dashes and solid lines, respectively. Small dashed lines show the NOEs relating the aand @-protonsof Cys' to a b-proton of Pro'.

2-

ms NOESY spectrum in HzO (illustrated for the Thr3-Met13 segment in Fig. 1).The two sections on either side of Pro' (and Pro") could be connected by NOEs relating the protons on the proline ring to protons of the preceding and following c residues. A strong NOE was observed between the a-H of : jj .: ....: j Cys7 (and Met13) and a 6-H of Proa (and Pro1') (illustrated for Cys7-ProSin Fig. 2), indicating a trans peptide bond for Cys7-Pros(and Met13-Pro14).Thea-H of Pro' (and was connected by NOE to the amide proton of Ile9 (and Led5) (illustrated for Proa-Ilegin Fig. 1).Intraresidual NOES were observed between all amide protons and theirown CB-protons, except for residues Aspz3and SerZ4 in the Thr3-Arg5segment. Several important long-range NOEs were observed in the 125ms NOESY spectrum in DzO (Fig. 3). A strong NOEwas observed between the Phe" 6-H andthe Ile9 a-H. More importantly, the t- and {"protons of Phe" showed definite NOEs to both @-methyleneprotons of Cys7 and Cys". Also, one of the 0-methylene protons of Cys7 showedNOEs to both 0-methylene protons ofCys". Chemical shifts of assigned resonances are shown in Table I. Relevant NOE data are summarized in Table 11. Only a few minor NOE cross-peaks remain unassigned, some of which may be due to impurities in the sample. Data Interpretation-An equilibrium situation with multiple conformations is typically encountered with short linear peptides in solution. However, the 6-kDa fragment of Fn, a FIG. 1. Backbone amide and aromatic region of a 960 ms dimer containing 52 residues, is roughly the size of bovine NOESY spectrum of the 6-kDa fibronectin fragment. Spectra pancreatic trypsin inhibitor. The size of this fragment, the were obtained from a 1 mM solution in 95% HzO and 5%D20 at pH fact that it is a dimer, and the presence of two interchain 7.0 and 25". The dNN connectivity pathways for residues Thr3-Cys' disulfide bonds, are expected to lend some conformational (solid lines) and Iles-Met13(large dashed lines) are indicated in the rigidity to its structure in solution. Comparison of 'H-chemibottom panel. Note that proline breaks the dNNconnectivity pathway a t 8 and 14. NOE contacts between aromatic and amide protons of cal shifts (TabIe I) of the Thr' toAr? segment with those of Phe" are shown with small dashes. Small dashed lines are also used random coil structures (Bundi and Wuthrich, 1979) shows to show the NOE contacts relating the aromatic protons of Phe" in that several resonances within the Thr3-Pro" sequence have the bottom panel, to the CB-protonsof Cys', Cys", and Phe", and to chemical shifts sufficiently different from random coil structhe C,-proton of Ile9 in the top panel. The a N cross-peak relating tures to indicate definite conformational preference in this Pro' and Ile' is shown in the top panel,along with the long-range do^ connectivities for (9-11) and (10-13). The &protons of residues 4,5, segment containing the inter-chain disulfide bonds. Consist6, 7, 9, 10, 11, 12, and 13 have been labeled in the top panel so as to ent with this observation, examination of Table I1 shows that inter-residue NOEs other than those showing daN, d m , dbN show the presence of intraresidual N,@;cross-peaks. 3-

!

8583

NMR-based 3 - 0 Structure of Fibronectin C-terminal Fragment

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

PPm FIG. 3. NOESY spectrum of the 6-kDa fragment in DSO. The COSY connectivity of the Phe" ring protons is shown in an insert at the bottom-left corner. The mixing time in the NOESY experiment was 125 ms. Other experimental conditions are as in Fig. 1. Long-range NOES relating the 8-protons of Cys7and Cys", and the Ile9 a-proton to thePhe" ring protons are shown by dashed lines. Solid lines are used to show NOES relating the C,- and CB-protonsof Cys7,Cys", and Ile'.

TABLEI 'H-chemical shifts for the assigned resonances of the C-terminal fragment at pH 7.0. 25°C Residue

NH

C.H

CSH

Other

~ C H 1.29 B Thr' 3.88 4.15 4.86 2.88, 2.77 Asn' 7CH3 1.17 4.32 8.30 Thr3 4.23 7NHZ 7.58, 6.94 4.73 8.44 2.86, 2.79 Asn' 4.09 7CH3 0.91, 0.91 8.05 2.09 Val6 4.77 7NHz 7.61, 6.92 8.49 Asn' 2.81, 2.72 5.04 8.27 CYST 3.08, 2.85 Pro' 4.53 2.46, 2.19 7CHz 2.08, 2.08; 6CHZ 3.84, 3.80 8.46 11.2 4.09 1.96 7CHz 1.53, 1.39; 7CH3 1.03; 6CH3 0.96 Glu" 7CHz 2.39, 2.27 9.02 4.16 2.19, 2.04 8.14 4.41 Cys" 2.58, 2.48 7.74 4.77 Phe" 6H 7.30; cH 7.12; {H 7.16 3.38, 2.96 Met13 7.81 4.81 2.11, 1.96 7CHz 2.61, 2.61; cCH3 2.12 Pro" 4.46 2.31, 1.95 7CHz 1.96, 1.96; 6CHz 3.80, 3.67 8.33 Leu" 4.32 1.64, 1.59 7 H 1.69, 6CH3 0.94, 0.88 8.39 4.61" Asp'6 2.79, 2.69 7.99 4.11 va117 7CH3 0.94, 0.94 2.11 8.38 Gln" 4.32 2.11, 1.99 7CHz 2.37, 2.37 8.24 ~1~19 4.30 1.40 Aspz0 8.32 4.61" 2.79, 2.70 Arg21 8.21 4.37 7CHZ 1.63, 1.63; KHz 3.22, 3.22 1.90, 1.77 G1uz2 8.45 4.31 2.10", 1.94 Aspz3 8.39 4.61" 2.79", 2.69" SerU 8.58 4.53 4.32, 4.23 Ar$6 1.80 1.92, 4.39 8.26 7CHz 1.63; 1.63, 6CHz 3.22 3.22, a The uncertainty (20.02 ppm) in these chemical shift values is larger than the uncertainty (f0.01 ppm) in the rest.

connectivities (Table 11, column 6) are observed only within this segment. For proline-containing peptides, the presence of multiple conformers is often indicated by the observation of two distinct sets of resonances corresponding to cis- and trans-prolines, since the rateof exchange between the species containing the two isomers is slow on the NMR time scale (Wuthrich, 1976; Larive et al., 1992). Resonances corresponding to only the trans conformers were observed for both Pro8 and in the 6-kDa Fn fragment, confirmed by the presence of strong NOE cross-peaks for both prolines. Based on these observations, it was assumed that a substantial population of the conformers of the 6-kDa fragment in solution had a preferred conformation for the Thr3-Pro14segment,

the structurebeing more flexible further away from the interchain disulfide bonds. Calculation of the three-dimensional structure, therefore, was attempted only for the Thr3-Pro14 segment. This was found to be more than adequate to demonstrate that the two interchain disulfide bonds linked the Fn monomers in an antiparallel fashion in the 6-kDa carboxyl-terminal dimer fragment. The NMR information indicates a symmetrical structure: chemical shiftsare identical for the same residue in both chains.Thispotentially complicates the interpretation of NOE data, since no distinction can be made between intrachain andinterchain NOES. Hence, a cross-peak relating residues i and i+l through space, might not necessarily indi-

8584

NMR-based 3 - 0 Structure of I Tibronectin C-terminal Fragment

TABLEI1 Summary of NOE information for the C-terminal fragment

of aH-NH, NH-NH, and pH-NH NOE cross-peaks (using a proportionality constant derived from the volume integrals of Residue dNBndmNbdNNb deNb Othef NOEs the crqss-peaks relating 6H and tH of Phe” across a distance of 2.5 A). Hence, the simplest interpretation of the NOE data Thr3 + + + in Table 11, is that all NOEs showing connectivities between Asn‘ + + ? + + Val’ + + + + main chain protons, and between main chain and Cg-protons Asn‘ ++ ? + (i.e. d u N , d N N , d g N ) represent intrachain sequential connectivCys’ ++- - ities. The statistical analysis noted above indicates that this should constitute afairly accurate representation of the threedimensional structure. This interpretation was used initially in both the deterministic and the probabilistic analyses. BePro’ - + - + 11~9 + + + cause of the observed chemical shift symmetry, the set of Glu” + + + + + sequential NOE connectivities were assumed to apply equally Cys” ++ + + to bothchains in the dimer. All d N g connectivities were assumed to be intraresidual, and hence intrachain only. Phe” ++ ? + The ambiguity concerning intra- versus interchain NOEs + + Met13 + - + arises solely due to the fact that the dimer structure is symPro1‘ Leu” ++ + + metric; otherwise residues n and n’ (i.e. the same residue on Asp” + + + + + the two chains of the dimer) would bedistinguishable by their ~ ~ 1 1 + 7 + + ? different chemical shifts. If only intrachain symmetry is asGln” + + ? + + sumed then the assumption that all “sequential” NOEs are ~ 1 ~ 1 + 9 ? + + intrachain is valid. On the other hand, if even a few of these Asp2’ + + + + ? sequential NOEs are interchain, then the identical chemical Argl + + ? + ? G1uZ2 + + ? + ? shift requirement imposes interchain symmetry in addition AspW ? ? ? + ? to intrachain symmetry. In other words, if the d,N connectivSeP‘ ? ? ? + ? ity observed between residues n and n + l (and hence between ArP + + ? ? ? n’ and ( n + l ) ’ ) , is interpreted as an interchain connectivity ’Intra-residual NOE: dNg (i,i);+ denotes NOE to the single @ between n and ( n + l ) ’ , the chemical shift symmetry requires proton for these residues;++ denotes NOE to both @ protons. that there must be a d,N connectivity between n’ and n + l . ‘Sequential connectivities: d(i,i+l). + and blank spaces, respectively, indicate the presence and absence of NOES, - denotes the This additional interchain symmetry would place more strinabsence of a proton in the residue (e.g. NHin Pro), such that the gent constraints on the structure. Also, from consideration of particular NOE cannot be present. “?” indicates the absence of NOE steric hindrancealone, it appearsunlikely that theinterchain information (e.g. due to bleaching of cross-peaks caused by water symmetry requirement could be satisfied in any arrangement suppression using presaturation). Insuchcases,bothpossibilities of the dimer structure. This is supported by the results of a (presence and absence of thecorresponding d connectivity) were set of calculations using conformational analysis alone (deconsidered, and two regionsin 4,$ space were predicted by FISINOE scribed under “Conformational Analysis”), without any con(see Table 3). sideration of the NOE data. e dw(i,j) are not repeated again as d-(j,i). While these NOES are For the deterministic approach, both intra- and interchain represented here as intrachain only (i.e. involving residues ( i , j ) ) ,the data does not exclude interchain NOES (i.e. those involving residues distance constraints were initially included for all NOE con(i,j‘)); bothpossibilities are consideredintheanalysis (see text). nectivities involving side chain protons. Later, during the Subscripts(@’,p”)and (b’,b”) have been usedto representthe two Cg- process of minimization of the penalty function for distance andCQ-protons (column 6) differentiating them in terms of their chemical shifts only (see Table l), and do not represent stereospecific constraints, those particular constraints that contributed to assignments, which can be obtained from the xI,x2values in Table large violations were gradually removed in an attempt to minimize the distance violations. 111. For the probabilistic approach, the sequential NOEs were cate a sequential connectivity, and could, in principle, repre- the only NOEs used. Since distance constraintswere not used sent along-range NOE cross-peak relating residues i on chain explicitly in this method, building the spatial structure did 1 and (i+1)’ on chain 2 of the dimer. However, a statistical not require that any prior decisions be made regarding analysis of short proton-proton distances in a collection of whether the other observed NOEs (involving side chain prodata from high resolution protein crystal structures (Billete? tons) were intra- or interchain NOEs. All unconstrained energy-refined structures were carefully examined to check et al., 1982) shows that 88% of the aH-NHdistances 5 3.0 A for consistency with experimentally observed NOEs. During represent sequential connectivities. The corresponding probthis examination, both intra- and interchain distances were abilities for NH-NH and pH-NH are 88 and 76%, respectively. checked to ensure that theselected structures were consistent The probabilities are even higher when such short interproton with at least the minimum number of experimentally observed distance limits are imposed simultaneously: the probaplity NOEs involving side chain protons in the particular dimer that the copnectivities are sequential when ( d a N 2 3.6 A and fragment in question. dNN 3.0 A) is 99%; it is 95% when ( d a N 5 3.6 A and d o N S: Structure Determination Assuming All Sequential ConnecIn other tivities to be Intrachain-The set of 55 approximate interpro3.4 A) and 90%when ( d N N 5 3.0 and d g N 5 3.0 words, protein folding patterns in nature very rarely lead to ton distance constraints per chain, derived from the observed short proton-proton distances relating main chain and Cg- NOE data, was used both with, and without the additional protons of residues that are not immediate neighbors on a symmetry constraint, to calculate three-dimensional strucpolypeptide chain. This conclusion was applied to the NOE tures of the Thr3 toPro14 segment, with the specific purpose data from the 6-kDa fragment, although the uniqueness of of determining whether the NMR data indicated a parallel or the interchain disulfide bonds makes it an unusual case. A an antiparallel arrangement of the two chains linked by the conservative estimate of the upper limit for the observed d a N , two interchain disulfide bonds. dNN,dgNconnectivities is 3.3 A, based on the estimation of The Deterministic Approach-Themore commonly used distances from the measured volume integrals for a sampling DG approach, followed by energy refinement, was used first.

A

A).

NMR-based 3 - 0 Structure of Fibronectin C-terminal Fragment

8585

TABLEI11 Several DG, DGREM, and DGRMD structures of the Thr3Comparison of re-estimated and final conformations M e P segment were obtained, all of which roughly satisfied the NOE constraints within this dimer fragment (with small Residue total distance violations).The structuresdid not fall into any -50,-55 g- g-58,-44 -55 S Thr3 closely related sets, and showed large variations in the main -65,-35 t,t t,t -54,-50 -175,-167 R Asn4 g-,t -75,-15 K chain conformation when compared in pairs. Addition of -75,-15 t t -56,-41 180 K Val' symmetry as a constraint in the energy refinement also gave -85,-15 t,t g-,t Asn6 Q numerous possible solutions (with identical monomer conforT -85,20 g-,t -60,173 -60,-40 mations in the dimer), again with a wide variation in the main -100,135g- g-65,158 -52 B cy2 chain conformation, as described above. This approach t showed that "constrained structures, roughly satisfying all-73,166Pro' A -50,160 -85,20 T t,t t,t -54,-41 -178,60 11~9 NOE requirements, were possible for both the parallel and t,g+ t,g+ the antiparallel dimer forms. However, all of the structures g-,g- g-kcalculated in this way contained several dihedral angles well g->t g-,t outside the sterically allowed regions. Therefore, comparison K -75,-15 t,t g-,t -47,-50 -66,-68 Glu" of the calculated conformational energies was not an adequate t,g+ g-,gcriterion, eitherfor selecting a setof preferred structures from g-&the many converged constrained structures, or for deciding g-, t -65,-35 g- g-57,-39 -62 Cys" R whether the NMR data indicated a parallel or an antiparallel t arrangement of the two chains in the dimer. We concluded t,p -57,-42 -64,70 -85,-15 g-,p Phe" Q that the number of constraints available was not sufficient T g-,p -85,20 for this strategy to work. -100,135 t,t g-,t -66,158 -63,-178 Met13 B The Probabilistic Approach-We then applied the BUILD t&?+ g-,gprocedure described above, using the d connectivities shown g-,gg-,t in Table 11 ( d , ~ ,dNN, d@N) to estimate corresponding regions -78,171 B --50.145 in d,$ space for each residue, shown in Table I11 (column 2). a Region denotes areas in $,$ space that correspond to particular The most probable @,$values corresponding to each region (Table 111, column 3) were used as the starting set of C$ and $ combinations of sequential d connectivities and are represented by a angles for the 12-residue monomer segment, Thr3-ProI4.The set of most probable b,$ values (Sherman et al., 1987). For Asn', and Phe" the NOE data indicates two regions in b,$ space. d,N connectivity data for three of these 12 residues, (Am4, Am6, Both regions are shown in column 2 for each of these residues. Column A d , and Phe"), are ambiguous, permitting two possible 3, therefore, contains two sets of the most probable b,$ values (in regions in d,$ space (see Table 111, column 2). However, the degrees) corresponding to these two regions. * Initial conformations. The initial x1 and x2 values are indicated two possible regions are close to each other in conformational space, and an average of the most probable C$,$values corre- as rotamers g+, g-, t, and p , representing 60, -60, 180, and go", sponding to thetwo regions was used as the startingdihedrals respectively. The IUPAC-IUB conventions (Hoffmann-Ostenhof, 1974) werefollowed in naming the torsion angles. (For valine, it in these cases. Extension of this method, and consideration should be noted that these conventions are different from those used of the intraresidual NOE data between amide- and Co-protons in CHARMM and INSIGHT.) was used to obtain the possible combinations of x1 and xz Final conformation. Column 5 shows the rotamers describing the angles for the side chain conformations (Sherman and John- various side chain conformations, including the energetically indistinson, 1991; Sherman and Johnson, 1993). A four-step "build- guishable set of conformations for the side chains of Ile', Glu'', and up" procedure wasfollowed to construct the final three- Met13 that lead to the set of 16 energy refined unconstrained strucconsistent with the NMR data. Values of b,$ and x1,x2(in dimensional structure: (i) TheCys7-Cys" segment, containing tures degrees) shown in columns 6 and 7 are for one of the 16 conformations the interchain disulfide bonds was constructed first, because (see text for angular root mean square deviations for pairs of these of the four long-range NOE restrictions between the Co- 16 structures). protons of Cys7 and Cys" (see Table 11) present in this segment. The Ile9 and Glu" side chains were initially trun- tion of parallel versus antiparallel arrangement of the monocated to alanine, making the testing sequence Cys-Pro-Ala- mers in the dimer fragment was answered at the first phase Ala-Cys. (ii) Phe" was added to this sequence, and, in two of the calculations in step (i), using the Cys7-Cys" segment subsequent steps, alaninesat positions 9 and10 were replaced only. A summary of the results for this stepof the calculations, byIle9 and GlulO. (iii) The Thr3-Asn6 segment was then using both parallel and antiparallel arrangementsof the monadded. As in (i) and (ii), calculations were first performed omer segment Cys-Pro-Ala-Ala-Cys to form the dimer, is with A m 4 and Am6 replaced by alanine. (iv) Met13 and given in Table IV. Of the four possible conformations correwere finally added to complete the segment. This procedure sponding to different combinations of x1 values for the pair reduced the total number of calculations required from 4096 of cystines in the monomer, none satisfied all four long-range X 2 (for parallel and antiparallel structures) to only 90. Proa and Prol4 were modeled to be in the trans configuration, as NOE requirements between the CB-protonsof Cys7and Cys" indicated by the presence of strong NOE cross-peaks (considering both intra- and interchain connectivities) for a parallel dimer structure. Two of the parallel structures (see (Table 11). The dihedral angles estimated by the FISINOE program rows i and ii in Table IV) failed to satisfy all sequential NOE were used as the startingangular coordinates. The Cys7-Cys" requirements, as well. This is apparent in the large deviations segment was built using the interactive graphics package, (Table IV) in backbone conformation from that estimated INSIGHT. CHARMM was then used to "patch" two such using sequential NOE data in FISINOE. The use of distance segments in either a parallel or an antiparallel fashion, via constraints, in addition to dihedral constraints, also did not the two interchain disulfide bonds. CHARMM was also used lead to anyunconstrained parallel dimer structures consistent for energy minimization, with gradual reduction, and finally with all NOE data. In the antiparallel dimer structure, it was elimination, of force constants for dihedral constraints, as possible to select one of the four combinations of xl conformdescribed under "Experimental Procedures." ers for the pair of cystines in the monomer (with x1 = g- = Arrangement of Monomer Chains in the Dimer-The ques- -60" for both Cys7 and Cys"; see row 1 in Table IV), since

8586

NMR-based 3 - 0 Structure of Fibronectin C-terminal Fragment TABLEIV

x1 angle for

each of the 2 cystines per monomer (Cys7 and Cys") may take three values (g- = -60", t = 180 "C, g+ = Conformational 60"),this leads to a total of 108 x 2 structures (for parallel armsdb Maximum Number of Cys"Cys' energ4 deviation' long-range and antiparallel forms). As a preproline residue, Cys7 was in + NOEs x1 x1 energies restricted to the/3 conformation. The conformational r = l c = 4 r=10 in + ant.iafintld calculated (using CHARMM) for the unconstrained struckcal/ml degrees tures were compared with thatof the structure in the@/3acua A. Monomers linked in an antiparallel fashion in the dimer -232.3 -48.5 -19.8 conformation, with x1 = -60" or g- for both Cys' and Cys". 23 17 4 1' g- gg-220.2 -45.9 -13.1 2 t When a structure witha comparable energywas obtained, the 42 85 2 Of 4 t -218.4 -42.3 -10.5 72 35 2 Of 4 3 gconformation of each residue was examined to see which of t -214.2 -44.7 -14.5 4 t 11 16 None thesequential d connectivities (previously assumedto be B. Monomers linked in a parallel fashion inthe dimer intrachain) were not present. Interproton distance calcula95 None i g- g- -236.4 -50.2 -16.1 46 tions were then performed, using the coordinates of the eng-235.8 -50.0 -15.7 ii t 42 97 None ergy-refined unconstrained structure, to check whether the t -215.3 -48.2 -19.5 30 18 1 of 4 iii gcorresponding interchain connectivitieswere present instead, -195.2 -33.4 -4.8 t iv t 13 22 1 of 4 in order to satisfy the NOE requirements. A summary of the a Energies calculated using values of 1, 4, and 10 for the dielectric results are given in Table V. Eight structures with conforconstant, t. mational energies comparable to the P@aaa(g-,g-) conforInitial backbone conformationsestimated using FISINOEare compared with conformations in the unconstrained, energy refined mation (see Table V, row 1) were found. Inall of these structures. The angular root mean square deviations (armsd) for $ structures,themonomers were linkedintheantiparallel only are shown, since armsd values for $J are similar foralleight fashion to form the dimer, and all retained conformational structures (about 23"). symmetryintheunconstrainedstructures (i.e. contained "The maximumdeviationin $ from estimated values has been included to show that some low energyconformations (see rows i and identicalconformations for the two monomers). However, interchain d connectivities (in place of missing intrachain ii in B) correspond to backbone dihedrals that have deviated more than 60" from the initial estimates consistent with sequential NOEs sequential d connectivities) were not found in any of these (or more than 30, where the armsd, o = 20", for FISINOE estimates structures, so that none of them satisfied all NOE requirements. of ($,$) in each region).This, in turn, implies that the corresponding (It is important to note here that ainfew of these structures, unconstrained structures do not satisfy allof the sequential NOEs. Cys7and Cys" could be satisfied Addition of distance constraints to satisfy long-rangeNOEs re- the long range NOEs relating by intrachain, rather than interchain connectivities. Thereof backbonedihedralsfromFISINOE sultedinlargedeviations of sequential NOE requirements. fore, whether the structure is parallel or antiparallel, it cannot estimated values, implying violation When both long-rangeand sequentialNOES were included as distance simply be assumed that NOEs relating Cys7 and Cys" must constraints, the resulting structures contained backbone dihedrals in be interchain.) Conformationalenergies for all parallel dimer sterically forbidden regions. structures were much higher than that obtained previously, e The single structure satisfying all sequential and long-range NOE assuming all sequential connectivities t o be intrachain (see requirements. Table IV). Interchain d connectivities (in place of missing intrachain sequentiald connectivities) were not found in any only this conformation satisfied all sequential and long-range NOE requirements. One interesting difference between the TABLEV parallel and antiparallel structures was that the symmetry Summary of results for the Cys-Pro-Ala-Ala-Cys sequence with requirement was satisfied, even in the absenceof any explicit symmetry constraints, in all four of the unconstrained anti- conformations other than that predicted by the assumption of intrachain sequential NOEs, with monomers linked in antiparallel fashion parallel structures, but in none of the parallel structures. i n the dimer a priori information Since the probabilistic approach uses Initial Conformational regarding empirical distribution functionsfor backbone conconformation energ4 armsdb Maximum Number Of for the CYS7 CYS" deviationb in formations in addition to the NMR data, the availableNOE NOEs CPAAC x1 x1 4 = 10 ( h +) in (@,+) satisfied data were sufficient for this method to distinguish between backbone parallel and antiparallel structures. kcallmol degrees Conformational Analysis-Since the results above were ob4 13,28 tained assuming all daN, dNN, dSN,and dNg connectivities to be1' p@aaa g- g- -48.5 -19.8 9,20 4 13,24 g' g- -46.3 -19.2 10,18 sequence, con- 2 @Baa. intrachain for the Cys7-Pro8-Alag-Ala10-Cys11 4 -47.4 -19.6 9,lO 14,20 @aaaa 3 g- t formational analysiswas performed for this sequence t o check 4 t 24,28 4 @aaaa g- -40.3 -19.8 14,14 whether other conformations (not predicted by the assumed 5 @aaaa g' g+ -54.7 -24.2 24,28 52,56 2 Of 4 2 of 4 intrachain sequential d connectivity patterns) were energeti- 6 Baaaa g+ g- -55.8 -20.6 13,19 24,25 4 15,35 g- g- -49.5 -18.1 9,26 cally favorable for this dimer fragment containing interchain 7 pppaa 2 Of 4 disulfidebonds. For simplicity,only twomain classes of 8 fla&x~ g+ g+ -45.8 -17.7 18,11 28,17 p a p ~ l g+ ~ l g-51.1 -19.8 5,29 7,40 None 9 backbone conformations were considered for each residue in "Energies calculates using values of 4 and 10 for the dielectric creating the starting structures: (i) the twisted or (Y conforconstant, c. mation, with (4,+) = (-60, -60); and (ii) the extended or /3 Initial backbone conformations used are compared with conforconformation, with (d,+) = (-60, 135). Since the actual semations in the unconstrained, energy refined structures (where the quence has a charged residue in position 10 (Glu"), the (60, initial values of ($J,$) for twisted (a)and extended (0)conformations 60) conformation (or a ~ )on , the right side of the (d,+) map, are assumed to be (-60", -60") and (-60",135")). The angular root was also considered for Ala". The structure obtained above, mean square deviations (armsd) and maximum deviation from the by assuming all sequential d connectivities t o be intrachain, initial values of $J and $ (columns 7 and 8) have been evaluated in may be represented as the /3/3a(~a( g-,g-) conformation under order to estimate the "goodness of fit" of the unconstrained structures initial conformations. this notation, withx1 angles for both Cys7 and Cys" close to to the The "reference" structure, i.e. the structure obtained assumingall g- (see Table IV, row 1).In accordance with the requirement sequential d connectivities to be intrachain. Note that of the nine of chemical shift symmetry, identical starting conformations conformationsshown in this table only this conformation satisfies all were used for the two monomer chains. Considering that the NOE requirements. Summary of results for the Cys-Pro-Ala-Ala-Cys sequence in step (iJ

NMR-based 3 - 0 Structure of Fibronectin C-terminal Fragment

8587

of these structures; norwere all the long-range NOE require- Replacing thesecond alanine by Glu" led to 16 conformations (4 X 4), eight of which were eliminated by energy criteria. ments satisfied. Also, none of theseparalleldimerforms Similarly, extending thesequence by the Thr3-Asn' segment retainedconformationalsymmetryintheunconstrained structures (i.e. conformations for the two monomers were not led to 32 possible conformations for the Thr3-Phe" segment (8 x 2 x 2), 24 of which were eliminated by energy criteria. identical in the unconstrained structure), thus violating the identical chemical shift requirement. Therefore, use of the Addition of Met13-Pro14resulted in 32 possible conformations chemical shift symmetry constraint, with the helpof confor- (8 X 4 x I), 16 of which were selected, using energy criteria, to be the final set of structures consistent with the NMR mationalanalysis, was sufficient todemonstratethatthe monomers in the carboxyl-terminal dimer fragment of Fn data. Although symmetry was not used explicitly as a conmust be linked in an antiparallel fashion by the interchain straint in the energy minimization process, thesymmetry built into the backbone conformation at the start was largely disulfide bonds. retained in the final ofset energy-refined unconstrained strucAt this juncture, it is important to note that the observation were also of NOE contactbetween Cys' and Cys" did not,of itself, rule tures. Similarly, long-rangeNOErequirements found tobesatisfiedinthe finaI16 structures,and were out a parallel structure; we had to do extensive additional obtained as a by-product of energy refinement, without the conformationalanalysisto show that only an antiparallel process. structure satisfied all of the NOE constraints. The analysis use of distance constraints in the minimization conformations of Estimation of Precision-The backbone by An et al. (1992) noted the existence of a ROESY crosspeak between the Cys' H, and theCys" HBz,(our numbering) the set of 16 final structures were very similar, and were and stated that"a close distance between the 2 Cys residues indistinguishable by x' statistical criteria. The 16 structures unambiguously indicates an antiparallel" arrangement.How- consisted of combinations of side chain conformations forIle9 ever, the chemical shift symmetry makes it impossible to (tt, tg', g g - , g-t), Glu'O (g-t, g-g-) and Met13 (g-t, g-g-1 that distinguish whether this ROESY cross-peak is intrachain or are consistent with the NMR data, and are indistinguishable interchain. Thus, the ROESY data alone, without any mod- by energy criteria. The rotamers representing these 16 side chain conformations are shown in Table 111, column 5 . The eling calculations, could not rule out a parallel structure in deviation (armsd)in backbone which the 2 cystines in the monomer are spatiallyclose, but angularrootmeansquare the disulfide bridge links Cys7-Cys7' and Cys"-Cys"', since conformations for pairs of structures within this set of 16 is ROESY cross-peaksbetween residues 7-7' and 11-11' cannot 6" for 4 and 10" for J/ (average of armsd values for all pairs within the 16 structures). The armsd for the estimatedvalues be observed due to the chemical shift symmetry. In fact, as we have shown above, it is quite possible to obtain a confor- of 4 and J/ in each region is about20" (Sherman and Johnson, mation in which Cys' and Cys" are close enough within the 1992). Table I11 also compares the dihedralangles estimated monomer to produce NOE contact. It is only the additional by the FISINOE program on the basisof the available NOE data, with the dihedrals in one of the 16 representative final modeling that demonstrates that this monomer conformation cannot then be coupled with a second monomer t o form a structures. The armsd between FISINOE estimates and calsymmetric dimer that satisfies all NOE constraints. Thus, the culated values of backbone dihedrals is 25" for 4 and 29" for analysis of An et al. (1992) assumed an antiparallel arrange- J/ (average of armsd valuesfor all 16 structures), and the ment, but did notprove it. estimated and calculatedbackbone structures are statistically ' criteria. Structure Consistent with NMR Data-At least nine differ- indistinguishable, usingx ent antiparallel dimer structures were found with comparable Unlike the types I, 11, and I11 repeat units of Fn, which containdominantstructuralfeaturesthatare common to conformational energiesfor theCys-Pro-Ala-Ala-Cyssequence, withfour different main chain conformations (~paacu, many proteins, the structureof the carboxyl-terminal dimer paaaa, pppaa, Papaa). However, as described in the above segment reported here is somewhat unusual.Fig. 4 shows the discussion onconformationalanalysis,onlyone of these conformation of the Thr3-Pro14 segment in the dimer form, (@@cuacu(g-,g-)in Table V, row 1) satisfied all NOE data: the with a single set of conformations for the side chains of Ile9, structure obtained assuming all sequential d connectivities t o Glu", and Met13 (as shown inTable 111). Thestructure Ile9be intrachain. Therefore, even if the Cys7-Cys" segment did consists of two twisted or coiled segments, Thr3-Asn6 and exhibit multiple conformations in solution, it appears likely Phe12, connected by an extended region at Cys7-Pro8. These that a substantial population exists in the ppaaa( g - g ) con- twisted regions may serve as recognition sites for the monoformation.Onlythisstructure was extendedto build the mers, and thus help to bring the2 cystines in each monomer three-dimensional structureof the Thr3-Pro14 segment. Intra- into close proximity, specifically in the antiparallel orientaresidual and sequential d connectivities shown in Table I1 tion, so that the required interchain disulfidebridges are were assumed to be intrachain in all subsequent calculations.formed in the correct manner in the dimer. The two ends of Only the antiparallel arrangement of the two chains was used, the Thr3-Pro14 segment are connected by interchain H-bonds with x1 = g- for both Cys7 and Cys". involving backbone atoms (NH of Am4 of one chain to 0 of On addition of Phe" to the Cys7-Cys" segment, only one Pro14 of the second chain). In addition to several inter- and of two possible conformations, with x1 -60" (or g-) and xz intrachain H-bondsinvolving sidechains, there arefour pairs = 90" (or p ) , for the PheIzside chain was found to satisfy the of intrachainH-bonds involvingbackbone atoms ineach long-range NOESbetween the CB-protonsof Cys7and the ring monomer. The donor-acceptor pairs are: (7, 3), (11, 8 ) , (12, protons of Phe" (taking into account both intra- and inter9), and (13,lO). The exposed surfaceof the two twisted regions chain connectivities). Since there were no strong long-range contain several hydrophobic side chains (Va15, Ile9, and Phe" NOE constraints relating the Cys'-Phe'' segment to the rest followed by Met13 and However, these may be covered of the structure, all subsequent calculations in the build-up by the rest of the monomer (LeuI5-G1uz6)folding back over procedure used the following energy criteria tochoose the set itself. The aromatic ringof Phe" in each monomerlies across of most probable conformations at each step: for a set of x1 the two disulfide bridges, and the hydrophobic interactions or xz rotamers, all conformations with energy greater than involving these aromatic side chains may help stabilize the the lowestenergy conformation by 4 kcal/mol(calculated disulfidebonds. The backbone structure of the Cys7-Cys" using a dielectric constant of lo), were eliminated. On replac- segment forms a loop that brings the 2 cystines in the same ing alanine by Ile9 in the Cys-Pro-Ala-Ala-Cys-Phe segment, chain (Cys7 and Cys") close enough to make intrachain difourenergetically equivalent conformations were obtained. sulfide bonds also possible. Why interchain and not intra-

8588

NMR-based 3-0Structure of Fibronectin C-terminal Fragment

FIG. 4. Stereo diagramof the ThrS-Pro'' dimer segment. The selected orientation shows the interchain disulfide bonds at the center of the figure. Hydrogens are not displayed. Residue numbers with primes belong to the second (symmetrical) chain. Ribbon traces have been added to illustrate the symmetry in the backbone conformation of the two chains. This figure represents one of the set of 16 structures consistent with the NMR data (see text).

chain disulfide bonds are observed in the 6-kDa fragment of Fn remains an intriguing question. Several x-ray protein structures areknown in which intrachain S-S bridges connect 2 cystines at i and i+4 positions (e.g. glyceraldehyde-3-phosphate dehydrogenase, insulin, lysozyme, proteinase (trypsin) inhibitor, ribonuclease T). The backbone conformation of this region in some of these structures is quite similar to that determined for the Cys7-Cys" segment of the 6-kDa fragment. It may be conjectured that hydrophobic interactions, such as those between Pro' and Phe" may hinder the formation of an intrachain disulfide bond inthe monomer, andthatthis hindrance is removed through stronger interchain hydrophobic interactions in the dimer. In all 16 final structures, the long-range NOEs relating the Cp-protons of Cys7 and Cys" were found to be interchain, while NOEs relating the Cpprotons of Cys' and ring protons of Phe" were found to be intrachain. A thorough understanding of the functions of Fn requires knowledge of the precise spatialarrangements of various binding domains and their interactions in the two similar subunits of the dimeric Fn molecule. OurNMR demonstration of the antiparallel arrangement of the interchain disulfide bridge near the carboxyl termini of Fn suggests that similar binding domains, such as the gelatin and cell-binding domains in different subunits, may be arranged in a diagonal manner rather thanin a mirror image. The present resultis consistent with previous work by Skorstengaard et al. (1986), which also suggested an antiparallel arrangement for the interchain disulfide bridge of Fn, based on HPLC patterns of peptides derived from the carboxyl-terminal 6-kDa fragment. The conformation of the Val5-Cys" region differs substantially from either of the average conformations proposed byAn et al. (1992). Several of the dihedral angles in the work of An et al. (1992) fall outside the usual sterically allowed regions. Since only the average conformations were reported byAn et al. (1992), we could not compare individual structures. There are significant differences between the observed NOE contacts for our work and those reported by An et al. (1992), thus the

structural differences reflect real differences in solution conformations, and notsimply different approaches to determining the structure. A detailed comparison suggests that truncation of the peptide chains at Val5and Cysll (our numbering, corresponding to their V1 and C7) permits substantially increased freedom for the disulfide-linked cyclic peptide ring due to loss of constraints from the extended peptide chain, and probably does notmaintain that fragment within its native environment andconformation. Thus, we propose that the structure determined here from a larger fragment of the carboxyl-terminal region is more likelyto represent the native conformation of the disulfide-linked segments of the carboxylterminal region of the two subunits. Fn is an essential component of the extracellular matrix that controls cell growth, cell shape, and differentiation (Hynes, 1990; Mosher, 1989). I n uitro, Fn molecules selfassemble into fibrils reminiscent of the fibrillar structures seen in thematrix (Vuentoet al., 1980).Although the detailed structure of the Fnfibrils is notknown, the current model for the assembly of the Fn fibrils, proposed by Hormann (1982), was based on a parallel interchain disulfide bridge pattern. According to thismodel, the fully extended form of Fn assembles into a half-staggered array to form 5-nm fibrils. Our determination of an antiparallel arrangement for the two Fn chains suggests that the fibril formation process may be far more intricate than presently perceived. It is of some interest to note that, relative to each other, the two chains extend away from the bridge region in a n essentiallyantiparallel fashion, in sharp contrast to the "folded," parallel arrangement suggested by An et al. (1992). Thus, one might speculate that thetwo chains may extend away from each other in fibril formation, although more extended structural information would clearly be required to definitively determine this arrangement. In conclusion, a three-dimensional structure has been determined for 24 residues of the dimer segment that contains the two interchain disulfide bonds within the 6-kDa carboxylterminal fragment of human plasma fibronectin. A build-up

NMR-based 3 - 0 Structure of Fibronectin C-terminal Fragment strategy for obtaining spatial structures was described, using the FISINOE method for evaluating local conformations from sequential d connectivities. The 2D NMR data are consistent with only an antiparallel arrangement of the two monomers connected via the two interchain disulfide bridges. Acknowledgments-We thank Dr. M. Prahhakaran for help and initial guidance in the use of the INSIGHT andCHARMM software packages, and for some initial calculations using DSPACE and CHARMM. REFERENCES Akiyama, S. K., and Yamada, K. M. (1987) Adu. Enzymol. Related Areas Mol. Biol. 6 9 , 1-57 An, S. A.A., Jimhez-Barbero, J., Petereson, T. E., and Llinas, M. (1992) Biochemistry 31,9927-9933 Baron, M., Norman, D., Willis, A., and Campbell, D. (1990) Nature 346,642646 Baron, M., Main, A. L., Driscoll, P. C., Mardon, H. J., Boyd, J., and Campbell, I. D. (1992) Biochemistry 3 1 , 2068-2073 Billeter, M., Braun, W., and Wuthrich, K (1982) J. Mol. Biol. 166,321-346 Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States,D. J., Swaminathan, S., and Karplus, M. (1983) J. Comput. Chem. 4 , 187-217 Bundi, A., and Wuthrich, K. (1979) Biopolymers 1 8 , 285-297 Constantine, K. L.,Brew, S. A., Ingham, K. C., and Llinas, M. (1992) Biochem. J. 283.247-254 Engvdl, E., and Ruoslahti, E. (1977) Int. J. Cancer 2 0 , l - 5 Garcia-Pardo, A,, Pearlstein, E., and Frangione, B. (1984) Biochem. Biophys. Res. Commun. 120,1015-1021 Hoffmann-Ostenhof, 0.(1974) Pure Ap 1. Chem. 40,291-308 Holland, S. K.. Harlos, K., and Blake, F. (1987) EMBO J . 6 , 1875-1880 Hormann, H. (1982) Klm. Wochenschr. 60,1265-1277

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Hynes, R. 0.(1990) Fibronectins, Springer-Verlag, New York Jeener, J., Meier, B. H., Bachman, P., and Ernst, R. R. (1979) J. Chem. Phys. 71.454fi-4.5.5.1 . ____ Kornblihtt, A. R., Umezawa, K., Vibe-Pedersen, K., and Baralle, F. E. (1985) EMBO J. 4,1755-1759 Larive, C. K., Guerra, L., and Rabenstein,D. L.(1992) J. Am. Chem. SOC.1 1 4 ,

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IdJl- I33 I

Leahy, D. J., Hendrickson, W. A., Aukhil, I., and Erickson, H. P. (1992) Science 268,987-991 Mosher, D. F. (ed) (1989) Fibronectin, Academic Press, New York Nerdal, W., Hare, D. R., and Reid, B. R. (1988) J. Mol. Biol. 2 0 1 , 717-739 Patthy, L. (1990) Semin. Thromb. Hemost. 16,245-259 Petersen, T. E., Thogersen, H. C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P., Sottrup-Jensen, L., and Magnusson, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 137-141 Sherman, S. A,, and Johnson, M. E. (1991) in Proteins: Structure, Dynamics and Design (Renugopalakrishnan, Carey, V. P. R., Smith, I. C. P., Huang, S. G., and Storer, A. C., eds) pp. 62-67, Escom Sci., Leiden, The Netherlands Sherman, S. A,, and Johnson, M. E. (1992) J. Magn. Reson. 96,457-472 Sherman, S. A., and Johnson, M. E. (1993) Prog. Biophys. Mol. Biol. 6 9 , 285339 Sherman, S. A,, Andrianov, A. M., and Akhrem, A. A. (1987) J. Biomol. Str. Dyn. 4,869-884 Sherman, S. A., Andrianov, A. M., and Akhrem, A.A. (1988) J . Biomol. Str. Dyn. 5 , 785-801 Skorstengaard,K., Jensen, M. S. Sahl P., Petersen, T. E., and Magnusson, S. (1986) Eur. J. Biochem. 161,441-463 States, D. J., Haberkorn, R. A,, and Ruben, D. J. (1982) J . Magn. Reson. 4 8 , 386-3x7 "-"-

Vuento, M., Salonen, E., Koskimies, A,, and Stenman, U. H. (1980) HoppeSeyler's 2.Physiol. Chem. 361,1453-1456 Wa er, G. (1983) Quart. Reo. Biophys. 1 6 , l - 5 7 W i g , G. Macura, S., Anil-Kumar, Ernst, R. R., and Wuthrich, K (1984) J . Magn. Reson. 66,207-234 Wuthrich K. (1989) Science 243,45-50 Wuthrich: K. (1976) NMR in Biological Research:Peptides and Proteins. North Holland, Amsterdam ~