Solution structure and dynamics of yeast elongin C in complex with a von hippel-lindau peptide1

doi:10.1006/jmbi.2001.4938 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 312, 177±186

Solution Structure and Dynamics of Yeast Elongin C in Complex with a von Hippel-Lindau Peptide Maria Victoria Botuyan1,2, Georges Mer2, Gwan-Su Yi1 Christopher M. Koth3, David A. Case2, Aled M. Edwards1,3 Walter J. Chazin2,4 and Cheryl H. Arrowsmith1* 1

Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto ON M5G 2M9, Canada 2

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla CA 92037, USA 3 Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto Toronto, ON M5G 1L6 Canada

Elongin is a transcription elongation factor that stimulates the rate of elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. It is heterotrimeric in mammals, consisting of elongins A, B and C subunits, and bears overall similarity to a class of E3 ubiquitin ligases known as SCF (Skp1-Cdc53 (cullin)-F-box) complexes. A subcomplex of elongins B and C is a target for negative regulation by the von Hippel-Lindau (VHL) tumor-suppressor protein. Elongin C from Saccharomyces cerevisiae, Elc1, exhibits high sequence similarity to mammalian elongin C. Using NMR spectroscopy we have determined the three-dimensional structure of Elc1 in complex with a human VHL peptide, VHL(157-171), representing the major Elc1 binding site. The bound VHL peptide is entirely helical. Elc1 utilizes two C-terminal helices and an intervening loop to form a binding groove that ®ts VHL(157-171). Chemical shift perturbation and dynamics analyses reveal that a global conformational change accompanies Elc1/VHL(157-171) complex formation. Moreover, the disappearance of conformational exchange phenomena on the microsecond to millisecond time scale within Elc1 upon VHL peptide binding suggests a role for slow internal motions in ligand recognition. # 2001 Academic Press

4

Departments of Biochemistry and Physics, and Center for Structural Biology, Vanderbilt University, 896 Preston Research Building, Nashville TN 37232, USA *Corresponding author

Keywords: elongin; von Hippel-Lindau; NMR solution structure; ligand recognition; conformational change

Introduction Mammalian elongin C was originally identi®ed as a member of the heterotrimeric transcription factor elongin; a complex that increases the rate of elongation of RNA polymerase II by suppression M.V.B. and G.M. contributed equally to this work. Abbreviations used: HSQC, heteronuclear singlequantum coherence; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; rmsd, root-mean-square deviation; VHL, von Hippel-Lindau; COSY, correlated spectroscopy; TOCSY, total COSY. E-mail address of the corresponding author: [email protected] 0022-2836/01/010177±10 $35.00/0

of transient pausing by that enzyme.1,2 In this complex consisting of elongins A, B and C, elongin C binds to and stimulates the activity of elongin A, the transcriptionally active subunit.3,4 The association of elongin C with elongin B further stabilizes the A/C complex. The subcomplex of elongin B and elongin C also binds to and stabilizes the product of the von Hippel-Lindau (VHL) tumor-suppressor gene.5 ± 7 The VHL gene is mutated in families with VHL disease, a genetic disorder that predisposes individuals to a variety of cancers including hemangioblastomas and renal clear-cell carcinomas.8 VHL binds to the elongin B/C complex via a motif that is mutated in >70 % of naturally occurring VHL mutants, strongly implicating the interaction with elongin in # 2001 Academic Press

178 its tumor-suppressor function.5,6,9,10 Elongins B and C also interact with proteins that bear homology to ubiquitin ligases9,11 ± 14 to form multiprotein complexes that exhibit ubiquitin ligase activity in vitro and in vivo.15 ± 17 The interactions of elongin C with VHL and elongin A depend on a short 12 amino acid residue consensus sequence termed the BC-box.3,6,18 The critical contact site on elongin C for elongin A and VHL resides in the C-terminal region,19 ± 22 but the details of how the elongin B/C/VHL complex interacts with other proteins, such as, Cul29,12 and Rbx123 are not known. Elongin C is conserved in sequence among mammals and other organisms such as Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae. Yeast elongin C (Elc1) exhibits 41 % identity and 71 % similarity with its mammalian counterpart.24 The greatest sequence similarity to mammalian elongin C spans residues Glu92 to Cys112, and includes the region that binds VHL and activates human elongin A.18 Elc1 was shown to bind both VHL and elongin A from mammalian sources and stimulate the latter in in vitro transcription assays.24 In yeast, an elongin A homologue (Ela1) is present but there is no obvious elongin B homologue.25 Previously, we have shown that free Elc1 in solution has regions of stable secondary structure, and that dynamic instability, most prominent at the C terminus, also exists.20 Addition of peptides corresponding to the BC-box of human VHL (residues 157-171) or yeast elongin A (residues 3-17) induces helix formation within both peptides and the C-terminal contact site of Elc1. Here, we present the three-dimensional solution structure of Elc1/VHL(157-171) complex. We also used chemical shift differences and previously published 15 N-NMR relaxation parameters26 to determine the extent of conformational and dynamic changes in Elc1 brought about by interaction with the VHL peptide. There are global changes in conformation and dynamics of Elc1 even though the peptideinteraction surface is limited to the two C-terminal helices. We postulate that this ``¯exibility'' in free Elc1 may be important for its ability to bind multiple proteins.

Results and Discussion General description of the Elc1 structure We previously reported that Elc1 by itself is dynamically unstable because a number of expected resonances were missing in the 15Nheteronuclear single-quantum coherence (HSQC) spectrum. We interpreted this as indicative of a subset of residues in the C-terminal region of the protein that have dynamic properties that broaden and obscure their NH resonances.20 However, upon addition of VHL(157-171) this dynamic instability was removed, and a complete set of resonances could be observed. In parallel, the

Structure and Dynamics of Elc1/VHL Peptide Complex

induction of an ordered structure from a random coil in the VHL(157-171) peptide can be inferred from the better dispersion of proton chemical shifts for the bound form of this peptide (data not shown). At all of the concentrations used for our NMR experiments, Elc1 and VHL(157-171) formed a 1:1 complex. Comparison of the 13C-®ltered, 12C-edited nuclear Overhauser enhancement spectroscopy (NOESY) spectra of 13C/15N-labeled Elc1 and unlabeled VHL(157-171), in the presence or absence of unlabeled Elc1 showed no differences (data not shown). This is a clear indication that there are no intermolecular contacts between molecules of the Elc1 protein, leading us to conclude that the protein is monomeric in the presence of VHL(157-171). Our previous sedimentation data suggested tetramerization of free Elc1 and dimerization in the presence of VHL(157-171). However, a detailed analysis by analytical ultracentrifugation under various conditions indicates that freshly prepared Elc1 is predominantly monomeric, and that higher-order complexes may be due to non-speci®c aggregation.26 The structure of VHL(157-171)bound Elc1 consists of a three-stranded b-sheet and four a-helices (Figure 1). The N-terminal b-strands 1 and 2 (Tyr5-Ser19) constitute a hairpin structure, while strand 3 (Arg43-Glu45) runs parallel with strand 2. Between strands 2 and 3 are two short a-helices (H1; Arg20-Ile25 and H2; Thr28Ile33) which are separated by a sharp turn involving a proline residue (Pro27). The transition from helix H2 to the third strand is an ill-de®ned loop (Glu34-Gly42). Helices H3 (Ser51-Ser68) and H4 (Ser87-Ile99) are at the C terminus, separated by a long loop, which is poorly de®ned from Gly69 to Pro78, and in an extended conformation from Glu79 to Ile82. The structural variations observed in the 32-42 and 69-79 residue segments re¯ect the high ¯exibility of these loops of Elc1 as the small angular order parameters Sang(f) in these regions correlate well with the small values of the Lipari-Szabo S2 order parameters for N-H vectors (vide infra), indicative of fast motion on the picosecond to nanosecond time-scale. The overall fold of Elc1 is very similar to that of the crystal structure of human elongin C in complex with human elongin B and VHL protein (B/ C/VHL)22 (Figure 1). The rmsd between the NMR structure of Elc1 closest to the average and the Ê when crystal structure of human elongin C is 2.1 A the two structures are automatically aligned on 88 Ca atoms with the program DALI.27 The ill-de®ned loop of Elc1 between helix H2 and the third b-strand corresponds to a region with no electron density in the crystal structure of B/C/VHL, indicating that this region is highly ¯exible in both structures. In the crystal structure the loop connecting helices H3 and H4 is well de®ned, with values of temperature factors similar to those found in regular secondary structure elements. This is in contrast to the corresponding region in the solution

Structure and Dynamics of Elc1/VHL Peptide Complex

179

Figure 1. Solution structure of Elc1/VHL(157-171). (a) Stereoview of the backbone (N, Ca, C0 ) of the 20 lowestenergy structures. The a-helices of Elc1 are blue and the b-sheet green. VHL(157-171) is orange. Each structure was Ê for the superimposed onto the average structure using residues 3-33, 42-68, and 80-97 of Elc1. The rmsd is 0.86 A Ê for all heavy atoms. (b) Ribbon representation66 of the structure closest to the backbone atoms N, Ca, C0 , and 1.71 A average using the same color coding as in (a). The helices (H) and b-strands (S) are indicated. (c) Ribbon representation of the crystal structure of human elongin C/VHL protein,22 regions corresponding to Elc1/VHL(157-171) are colored as in (a).

structure of Elc1, which is disordered and highly ¯exible, perhaps due to the insertion of three aspartic acid residues (73-75) in Elc1. Elc1 protein/VHL(157-171) peptide interface When bound to Elc1, VHL(157-171) is entirely helical. Changes in proton chemical shifts and the appearance of sequential amide nuclear Overhauser enhancement (NOE) connectivities upon protein binding were consistent with a helical structure, as seen in the crystal structure for the corresponding region of the VHL protein in complex with human elongins B and C.22 The proteinpeptide interface is highly hydrophobic (Figure 2). The C-terminal a-helices H3 and H4, and the loop connecting these two helices of Elc1 de®ne the binding groove for the peptide (Figure 3). The contact residues of Elc1 include Tyr60, Ile82, Thr84,

Ser87, Leu88, Leu90, Leu91 and Ala94, while those of the VHL peptide consist of Thr157, Leu158, Cys162, Leu163, Val165, Val166 and Leu169. Note that salt-bridges or hydrogen bonds may exist involving Lys159 from the peptide and Asp95 from Elc1, and Arg161 and Glu79 and/or Glu81 (Figure 3). All of the contact residues of VHL(157171) to Elc1 are conserved in sequences known to bind mammalian elongin C. Some of the important VHL residues at the interface, Thr157, Leu158, Cys162 and Val165, constitute a subset of the frequently mutated residues in patients with the VHL disease.28 ± 31 Mutations of these amino acids, except Val165, lead to impairment in elongin C binding. Similarly, these mutations in elongin A affect its normal activity as a result of disrupted binding to elongin C.18 Most interacting residues of yeast and human elongin C are conserved and maintain similar con-

180

Figure 2. Planes from the 3D 13C-edited, 13C-®ltered NOESY experiment showing intermolecular NOE correlations involving Ile82, Thr84 and Ala94 of Elc1, and several residues of VHL(157-171).

tacts with VHL(157-171):22 Tyr60, Ile82, Leu88, Leu90, Leu91 and Ala94 in Elc1 correspond to Tyr83, Ile95, Leu101, Leu103, Leu104 and Ala107, respectively, in human elongin C. Only Thr84 and Ser87 in Elc1 are not conserved but are replaced by Pro97 and Ala100, respectively, in human elongin C. In the crystal structure, hydrogen bonds are present between the side-chains of Lys159 and Asn108, and Arg161 and Glu92. Glu92 and Asn108 correspond to Glu79 and Asp95, respectively, in Elc1. Conformational change in Elc1 upon binding VHL(157-171) peptide Comparison of 1HN and 15N chemical shifts of free and VHL(157-171)-bound Elc1 reveals signi®cant differences.20 When these chemical shift

Structure and Dynamics of Elc1/VHL Peptide Complex

changes are mapped on the structure of the Elc1/ VHL(157-171) complex (Figure 4), the largest number of affected resonances is in the vicinity of the peptide-binding site, namely helices H3 and H4, and the intervening loop. As noted previously, in the absence of VHL(157-171) most of helix H4 resonances are missing due to exchange broadening, whereas they are all present after addition of peptide, indicative of a conformational change or stabilization of the binding site.20 The chemical shift perturbations propagate away from the binding region to the b-sheet and helices H1 and H2, which suggests an extensive change in the conformation of Elc1. Such interpretation of chemical shift variations in terms of a conformational change is supported by the previously reported large difference between the R1 backbone 15N-spin relaxation rates for free and VHL(157-171)-bound Elc1,26 which are likely to originate from very different hydrodynamic properties of peptidebound versus free Elc1. The markedly different dynamic behaviors are illustrated here by direct mapping of the spectral density functions J(o) for the backbone 15N-1H vectors of both free and peptide-bound Elc1 at 0 and oN frequencies (Figure 5). The reduced spectral density approach32 ± 35 was used in these calculations. A linear correlation is observed between J(0) and J(oN). Linear relationships between spectral density samplings at different frequencies have been noticed for other proteins, and exploited to extract correlation times.32,36,37 If the re-orientation of an internuclear 15 N-1H vector is a composite function of non-correlated motions, the corresponding spectral density function may be expressed by a linear combination of spectral density terms. Proceeding from this assumption and using the simplest form, a Lorentzian lineshape, for each spectral density term, one can derive a third-degree equation in t where the meaningful roots give the correlation times of the motions contributing to the low-frequency part of the spectral density function (equation (5) in Methods). Note that this methodology is applicable only in cases where the molecule tumbles isotropi-

Figure 3. Stereoview of Elc1/ VHL(157-171) interface. The protein part is represented in blue and the peptide in brown. The hydrogen atoms have been deleted from the displayed side-chains for clarity.

181

Structure and Dynamics of Elc1/VHL Peptide Complex

Figure 4. Chemical shift perturbations in Elc1 upon VHL(157-171) binding. Elc1 is shown in blue and VHL(157-171) in orange. Residues with combined chemical shift changes of backbone 1H and 15N resonances ([(
d1H)2 ‡ (
d15N)2]1/2) larger than 50 Hz are colored yellow. The C-terminal helix of Elc1, for which most amide resonances were missing in the absence of VHL(157-171), is shown in gray.

cally. Such analysis gives correlation times of 8.8 ns and 0.8 ns for free Elc1, and 16.0 ns and 0.7 ns for VHL(157-171)-bound Elc1. On the basis of their magnitude, the two values for free Elc1 may be assigned to the overall tumbling of the molecule and the internal motion. Notably, a correlation time of 8.8 ns is in agreement with the expected range for a globular protein of the size of Elc1 (12 kDa) at 25  C.36 The much larger value obtained for the complex (16.0 ns) cannot be accounted for by the moderate increase in molecular mass due to addition of peptide. In the absence of any aggregation in the sample,26 we interpret this as a conformational change leading to an anisotropic tumbling of Elc1/VHL(157-171) complex. Slow motions in the microsecond to millisecond range in free Elc1,20,26 combined with conformational change and stabilization upon interaction, may underlie the ability of Elc1 to bind several different proteins. The absence of BC-box in a number of putative Elc1 binding proteins38 suggests that Elc1 may also accommodate in its binding groove amino acid sequences other than the BC-box. Alternatively, there may be other binding region(s) in Elc1. A change in Elc1 conformation is also observed upon interaction with another BC-box-containing peptide. In the presence of Ela1(3-17), Elc1 assumes a conformation very

Figure 5. Plot of J(oN) as a function of J(0) for free Elc1 (black) and Elc1/VHL(157-171) complex (red). The ®ts to equation (5) were obtained by linear regression. The arrows point to the meaningful solutions of the equation. The resulting values for the apparent overall rotational correlation times are indicated. The broken curve represents the theoretical plot of J(oN) as a function of J(0) at various values of the correlation time for a spectral-density function with a Lorentzian lineshape. Points of free Elc1 with an abscissa value higher than the theoretical plot values, indicative of slow motions occurring on a microsecond to millisecond time-scale, were excluded from the ®t.

similar to that in the Elc1/VHL(157-171) complex, and the perturbed Elc1 resonances extend to residues beyond the peptide-binding site. The 15NHSQC spectra of Ela1(3-17)- and VHL(157-171)bound Elc1 can be superimposed.20 However, the conformational ¯exibility of Elc1 appears to be partly retained in the complex with Ela1(3-17), as resonances at the C terminus of Elc1 remain missing. Dynamics of Elc1/VHL(157-171) complex The backbone 15N-spin relaxation parameters R1 and R2, and 15N-(1H) steady-state nuclear Overhauser effect of free Elc1 and Elc1/VHL(157-171) complex were published by Buchberger and coworkers.26 As noted by these authors, and consistent with our spectral density mapping analysis (Figure 5), the variations observed in R1, with values ranging from 0.9 to 1.4 sÿ1, suggest that the rotational diffusion of the protein-peptide complex is anisotropic. With the structure of Elc1/ VHL(157-171) accessible we were able to estimate the rotational diffusion tensor, and use this information to perform a more quantitative analysis of the local motions of the polypeptide chain. For 1H-15N vectors undergoing low-amplitude rapid motions the R2:R1 ratios are essentially independent of internal motion and are de®ned by the overall tumbling of the molecule. If the three-

182 dimensional structure of the molecule is available, the R2:R1 ratios for rigid 1H-15N vectors can be used to derive the molecule's rotational diffusion tensor.39 ± 42 Thus, 23 residues from the b-sheet and a-helices of Elc1 were used to characterize the rotational diffusion tensor, constrained to an axially symmetric model. The orientation and magnitude of the principal components of the axially symmetric diffusion tensor are: Dk/ D? ˆ 1.55  0.37 (with Dk ˆ Dzz and D? ˆ Dxx ˆ Dyy), y ˆ 1.51  0.28 rad, and f ˆ 5.30  0.12 rad. The internal dynamics of Elc1/VHL(157-171) was then analyzed in terms of the model-free formalism43 ± 45 by ®tting the components of the axially symmetric rotational diffusion tensor and the relaxation parameters to ®ve dynamic models, as described by the authors of the Modelfree program.46 For each N-H vector, these ®ts give an estimate of the order parameter (S2), the correlation times (te) for fast internal motions, and a phenomenological chemical exchange contribution Rex (Figure 6). The data show that regions of the molecule with low angular order parameters Sang(f); (i.e. the N terminus, and loop segments 32-42 and 69-79) are in fact dynamically disordered, since there is a good correlation between vectors with low values of Sang(f) and S2 order parameters (Figure 6). Note that none of these segments in fast motion is directly involved in peptide binding. Perhaps the most striking observation is that only a small number of residues are involved in slow motion; only six residues best ®t a model with an Rex term. This is in contrast with the dynamics data available for free Elc1,20,26 which clearly indicate extensive conformational exchange on the microsecond to millisecond time-scale, especially in the peptide-binding region. There are several examples of proteins in which the binding residues participate in slow-exchange processes33,47 ± 54. It has been suggested that these slow-exchange phenomena are important for rapid protein-ligand binding, and may facilitate the recognition of multiple ligands. The fact that the slow conformational exchange in Elc1 backbone is not retained upon peptide binding stresses the importance of slow motions in the recognition process.

Materials and Methods Sample preparation 15

N- and 15N/13C-labeled Elc1 in complex with unlabeled VHL(157-171) were prepared as described.25 Brie¯y, Ela1(1-143) and hexahistidine-tagged Elc1 were co-expressed in Escherichia coli BL21 (DE3) cells grown in M9 medium containing 15NH4Cl or 15NH4Cl/[13C]glucose. The cells were lyzed and subjected to metal chelate, ion-exchange and gel-®ltration chromatography. Elc1 was isolated from the Ela1(1-143)/Elc1 complex by urea denaturation, and passage through a nickel resin that traps only Elc1. Subsequently, Elc1 was renatured by serial dialysis, and the histidine tag removed by throm-

Structure and Dynamics of Elc1/VHL Peptide Complex

Figure 6. Stuctural dynamics of Elc1/VHL(157-171).  angular order parameters (Sang(f)) calculated from the ensemble of 20 NMR structures and Modelfree parameters (S2, te and Rex) calculated using the NMR structure closest to the average and previously published 15N R1, R2 and (1H-15N) NOE data.26 Of the 60 nuclei analyzed, 35 were best ®t with model 1, one with model 2, six with model 3, and 16 with model 5.

bin digestion. Elc1 was then dialyzed against NMR buffer and concentrated to 0.3-1.5 mM by ultra®ltration. Aliquots of VHL(157-171) (NH2-TLKERCLQVVRSLVKCO2H) solution were added to Elc1 until equimolar amounts were attained. NMR spectroscopy All NMR spectra were recorded at 30  C on samples containing 0.3 to 1.5 mM Elc1 in complex with VHL(157171), 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 7.5 mM DTT dissolved in either 7 or 99.99 % 2 H2O. NMR experiments were performed on Varian Unity and Unity‡ and Bruker AMX and DRX spectrometers operating at 500, 600, 750 and 800 MHz proton frequencies. The 1H, 13C and 15N resonances of free and VHL(157-171)-bound Elc1 have been assigned.20 Only partial assignment of VHL(157-171) could be obtained because of the poor quality of the [F1-13C/15N, F2-13C/15N]-®ltered correlated spectroscopy (COSY) and total COSY (TOCSY) spectra.55,56 However, the observed changes in 1H chemical shifts of the peptide upon binding Elc1 were consistent with an a-helix, and the [F1-13C/15N, F2-13C/15N]-®ltered NOESY spectrum

183

Structure and Dynamics of Elc1/VHL Peptide Complex Table 1. Statistics of 20 NMR structures Distance restraint violations Ê Number >0.2 A Ê) Maximum NOE violation (A Torsion angle violations >5  Mean constraint violation energy (kcal molÿ1) Mean AMBER energy (kcal molÿ1) rmsd from ideal covalent geometry Ê) Bond length (A Bond angle (deg.) Ê) rmsd from the average structure (A Backbone heavy atoms (residues 3-33, 42-68 and 80-97) All atoms (residues 3-33, 42-68 and 80-97) Backbone stereochemistry (%) determined with PROCHECK for all residues of Elc1 and VHL(157-171) Most favorable regions Allowed regions Generously allowed regions Non-allowed regions

(tm ˆ 150 ms) showed sequential amide NOE connectivities also supporting a helical conformation. Interproton distances (216 intra-residue; 285 sequential; 169 mediumrange; 175 long-range, including eight intermolecular; and 371 ambiguous, including two intermolecular) were derived from 3D 15N-edited NOESY (tm ˆ 80 ms) and 3D 13C-edited NOESY (tm ˆ 100 ms). Intermolecular NOEs were assigned using a 3D 13C-edited, 13C-®ltered NOESY experiments (tm ˆ 150 ms).57 The 61 f dihedral angle restraints were obtained based on 3JHN-Ha coupling constants58 and 61 c restraints were assigned to residues for which Ca and Ha chemical shifts indicated helical conformations. Eighteen pairs of hydrogen-bond restraints were included based on the identi®cation of slowly exchanging amide protons and characteristic NOEs. The peptide f and c dihedral angles were constrained within the helical range. NMR spectra were processed and analyzed with the NMRPipe/NMRDraw59 and NMRView60 softwares.

Structure calculations One hundred structures of Elc1/VHL(157-171) complex were calculated with the program DIANA using the REDAC strategy with the unambiguous distance and dihedral angle restraints.61 In DIANA, the complex was treated in the same way as a single molecule using the ``invisible linker'' option to connect the peptide and protein sequences. The generated structures were re®ned by restrained molecular dynamics using AMBER with inclusion of the ambiguous NOEs.62 The calculation protocol consisted of 3000 steps of energy minimization prior to two high-temperature runs at 1000 K for 20 ps of restrained, simulated annealing. Cooling to 298 K proceeded for 14 ps. All distance restraints within the peptide and protein were maintained with a force constant Ê ÿ2. Initially, the force constants for of 20 kcal molÿ1 A trans-o, f and c were all set to 150 kcal molÿ1 radÿ2. For the ®nal annealing step f and c force constants were reduced to 50 kcal molÿ1 radÿ2. All DIANA and AMBER calculations were performed on SGI PowerChallenge or Origin 2000 computers. A ®nal representative ensemble of 20 structures of Elc1/VHL(157-171) was selected for analysis based on restraint violation energies.

19 0.27 0 ÿ1557.27 6.54 0.01 1.8 0.86 1.71 77.7 17.9 1.6 2.8

Analysis of relaxation data The spectral densities at 0, oN and oH ‡ oN frequencies (J(0), J(oN) and , respectively) were calculated from the published 15N R1, R2 and 15N-(1H) steady-state nuclear Overhauser effect under the assumption that the values of J(o) at high frequency are very similar.32 ± 35 The spectral densities were analyzed by plotting the values of J(oN) as a function of J(0). Linear correlations between spectral density samplings at different frequencies have been noted for several proteins32,36 and DNA oligomers.63 As proposed by LefeÁvre et al.,32 if one assumes that the various motions are not correlated, the observed linear correlation between J(oN) and J(0) may be expressed by a linear combination of spectral density terms characterizing each motion. Thus, from the linear relationship observed experimentally, we write: J…oN † ˆ aJ…0† ‡ b

…1†

where a and b are the slope and the intercept, respectively, of the ®t (Figure 5). Assuming that the spectral density function is a composite function re¯ecting noncorrelated motions, one may express J(o) as a weighted sum: X J…o† ˆ ai Ji …o† …2† i

where ai are the scaling factors of the spectral density functions characterizing each motion and iai ˆ 1. By substitution, equation (1) becomes: X X ai Ji …oN † ˆ a ai Ji …0† ‡ b …3† i

i

Similarly, each Ji(o) component should obey the linear relationship: Ji …oN † ˆ aJi …0† ‡ b

…4†

If the Ji(o) components are given a Lorentzian lineshape J(o) ˆ 2t/5(1 ‡ o2t2) with t being the correlation time, equation (4) leads to a third-degree equation in t: 2ao2N t3 ‡ 5bo2N t2 ‡ 2…a ÿ 1†t ‡ 5b ˆ 0

…5†

the meaningful roots of which give the correlation times.

184

Structure and Dynamics of Elc1/VHL Peptide Complex

The principal components of the axially symmetric diffusion tensor64 were estimated by ®tting the experimental and calculated R2:R1 ratios,39 ± 41 with the structure of Elc1/(VHL157-171) closest to the average as a reference. For these calculations, 23 residues de®ned as rigid were selected within the regular secondarystructure elements of Elc1. The following selection criteria were applied:39 for each residue n 15N(1H)NOE 5 0.65 and: hR1i=R1n ÿ hR2i=R2n 41:5  SD where the R1 and R2 averages are taken over residues that have not been excluded because of a low NOE and SD is the standard deviation calculated for those residues. The components of the axially symmetric diffusion tensor and relaxation parameters were then ®tted to ®ve Lipari-Szabo43,44 and extended Lipari-Szabo dynamic models45 using the Modelfree program (version 4.01). Model selection employed the statistical protocol described by the authors of Modelfree.46 The parameters adjusted during the ®tting process were: for model 1, S2 only; model 2, S2 and te; model 3, S2 and Rex; model 4, S2, te and Rex; model 5 (extended Lipari-Szabo), S2f , S2s and te, where S2f , S2s are the generalized order parameters for motions at two different time-scales.45 Using model 5 is analytically equivalent to a Lipari-Szabo-type analysis assuming a fully anisotropic rotational diffusion.65 Errors used in the analysis of relaxation data are those reported for 15N R1 and R2.26 An uncertainty range of 30 % was used for the 15N-(1H) steady-state nuclear Overhauser effect data. Uncertainties on the calculated values of the reduced spectral density functions were evaluated by performing 250 calculations with random Gaussian noise added to the relaxation rate constants. The width of each Gaussian distribution was set to the range of errors on the corresponding relaxation rate constant. Data Bank accession number The atomic coordinates for the best 20 re®ned structures have been deposited in the Protein Data Bank with the accession code 1HV2.

Acknowledgments We thank John Chung for help in the setup of NMR experiments; Adelinda Yee, Gerard Kroon, Micah Gearhart, Brendan Duggan and Signe Holmbeck for assistance with NMRPipe/NMRView; and Gerard Kroon for assistance with Modelfree. This research was supported by grants from the National Cancer Institute of Canada to C.H.A. and A.M.E, the National Science Foundation and Vanderbilt University Molecular Toxicology and Ingram Cancer Centers to W.J.C., and the National Institutes of Health (GM45811) to D.A.C.

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Edited by M. F. Summers (Received 10 January 2001; received in revised form 16 July 2001; accepted 16 July 2001)