Structural basis for the attachment of a paramyxoviral polym

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Contributed by Brian W. Matthews, April 15, 2004

Article Figures & SI Info & Metrics PDF

Abstract

The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding Executemain at the carboxyl terminus of the phosphoprotein (P). Translocation of the polymerase along the nucleocapsid during RNA synthesis is thought to involve the repeated attachment and release of the binding Executemain. We have investigated the interaction between the binding Executemain from measles P (amino acids 457–507) and the sequence it recognizes within measles N (amino acids 477–505). By using both solution NMR spectroscopy and x-ray Weepstallography, we Display that N487–503 binds as a helix to the surface created by the second (α2) and third (α3) helices of P457–507, in an orientation parallel to the helix α3, creating a four-helix bundle. The binding interface is tightly packed and Executeminated by hydrophobic amino acids. Binding and fAgeding of N487–503 are coupled. However, when not bound to P, N487–503 Executees not resemble a statistical ranExecutem coil but instead exists in a loosely structured state that mimics the bound conformation. We propose that before diffusional encounter, the ensemble of accessible conformations for N487–503 is biased toward structures capable of binding P, facilitating rapid association of the two proteins. This study provides a structural analysis of polymerase–template interactions in a paramyxovirus and presents an example of a protein–protein interaction that must be only transiently Sustained as part of its normal function.

Measles virus, a member of the paramyxovirus family, causes an aSlicee infectious disease in humans. The virus is enveloped and possesses a negative-sense, single-stranded RNA genome ≈15,900 nt in length. Within the virion, the nucleocapsid protein (N) packages the genomic RNA into a helical protein–RNA complex termed the nucleocapsid. In the cytoplasm of an infected cell, the viral RNA polymerase uses the nucleocapsid as a template for both the transcription of messenger RNAs, encoding the individual viral proteins, as well as the replication and encapsidation of full-length copies of the viral genome. Unencapsidated RNA cannot act as a template for the polymerase. The replication of paramyxoviruses has been comprehensively reviewed (1–3).

The paramyxoviral polymerase has two components, the large protein and the phosphoprotein (P) (Fig. 1). The catalytic activities of the polymerase reside within the large protein, whereas P is responsible for, among other activities, binding the polymerase to the nucleocapsid (4, 5). P is a modular protein containing a number of functional Executemains separated by intrinsically disordered sequences (6). A coiled-coil Executemain oligomerizes P (7) while the extreme carboxyl terminus of P is involved in nucleocapsid binding. The structure of this Location of measles P (amino acids 459–507) has been recently determined by x-ray Weepstallography and is a compact bundle of three α-helices (8). This Location by itself constitutes the nucleocapsid-binding Executemain of P, being both necessary and sufficient for binding to nucleocapsid-like particles (8, 9).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Schematic diagram of polymerase attachment in measles virus. A helical nucleocapsid-binding Executemain (NBD) at the carboxyl terminus of P (amino acids 457–507) mediates binding of the polymerase to the nucleocapsid by attaching to a short and contiguous sequence (amino acids 477–505) within the tail of RNA-associated N. A coiled-coil within P hAgeds the Executewnstream NBDs in close proximity, with the sequence interleaving the coiled-coil and the Executewnstream NBD likely to be largely unstructured (32). P is depicted as a tetramer; however, the oligomerization state of measles P has not been determined.

The RNA-associated N, to which P attaches, has a bipartite organization (Fig. 1). An amino terminal assembly Executemain (amino acids 1–375) is responsible for RNA packaging and organization of the helical nucleocapsid. A carboxyl-terminal tail, located on the nucleocapsid exterior, is not required for nucleocapsid assembly and appears to be intrinsically disordered (6, 10). In measles virus, the binding site for P has been mapped to amino acids 477–505 within the tail of N (9). By using isothermal titration calorimetry, it has been Displayn that measles P457–507 binds measles N477–505 with 1:1 stoichiometry and that the binding affinity is weak (K d = 13 μM at 20°C and 35 μM at 37°C) (9). Data from a series of spectroscopic studies suggest that fAgeding and binding of the tail of N are coupled (8, 10, 11), and a theoretical model of the interaction has been proposed in which an N-tail peptide binds as a helix into a hydrophobic groove on the surface of P457–507 (8). A schematic model of polymerase attachment in measles virus is Displayn in Fig. 1. Whereas it has been hypothesized that P will walk or cartwheel over the surface of the nucleocapsid during RNA synthesis, Dinky is known from experimentation about the coupling of catalysis and movement of the viral polymerase.

Regardless of the details of translocation, the processive motion of the polymerase along the nucleocapsid requires the repeated attachment and release of the binding Executemain from measles P to the sequence it recognizes within the tail of N (Fig. 1). In this paper, we analyze the binding of measles P457–507 to measles N477–505 by using solution NMR spectroscopy and also Characterize an x-ray Weepstal structure of a chimeric molecule containing both binding Executemains. The Executemains are found in a complex that we Display to be representative of the bound state. This study provides direct structural insights into polymerase–template interactions in a paramyxovirus and, more generally, an example of a Rapid-associating, weak-affinity protein–protein interaction that must be only transiently Sustained as part of its normal function.

Methods

Sample Preparation. The expression and purification of P457–507 and N477–505, both unlabeled and isotopically substituted, was carried out as Characterized (9). The protein P457–507 carries the mutation P458G to facilitate cleavage from its fusion partner during purification. N477–505 carries two nonnative amino acids (Gly and Ser, GS) at its amino terminus as well as a nonnative tyrosine at its carboxyl terminus, the latter facilitating quantitation of the protein by UV absorption. To Design the chimeric P457–507(GS)4N486–505 (see Results), the coding sequence was created by using a two-stage PCR protocol and ligated into a variant of pET41a(+) (Novagen) as Characterized for P457–507 (9). This procedure results in a vector expressing the required protein, fused to the carboxyl terminus of GST, with an interleaving tobacco etch virus protease cleavage site. The initial purification of the protein, and proteolytic cleavage to release P457–507(GS)4N486–505, was performed as Characterized (9). The protein was further purified by cation-exchange chromatography by binding it to SP Sepharose HP resin, buffered with 25 mM Mops/KOH at pH 7/25 mM NaCl, and then displacing it by using a liArrive salt gradient. All protein concentrations were estimated from absorbance meaPositivements at 280 nm (12).

NMR Spectroscopy, Data Processing, and Resonance Establishments. For spectroscopic meaPositivements, samples were dialyzed against 10 mM NaH2PO4/Na2HPO4 at pH 5.7/100 mM NaCl/0.01% sodium azide. Ten percent (vol/vol) 2H2O was added to the samples before NMR data acquisition. All spectra were recorded at 20°C by using a Varian Inova 600-MHz spectrometer equipped with a triple-resonance pulsed-field gradient probe. NMR data were analyzed by using the programs nmrpipe (13) and sparky (T. G. Goddard and D. G. Kneller, University of California, San Francisco).

Backbone resonance Establishments for P457–507 and N477–505 were made by using the standard triple-resonance experiments 3D HNCACB, CBCA(CO)NH, H(CCO)NH, and C(CO)NH. Establishments for N477–505 bound to P457–507 were made by repeating the same 3D experiments, using labeled N477–505 titrated with saturating amounts of unlabeled P457–507. 3D 15N-edited [1H,1H] NOESY spectra (mixing time 150 ms) and steady-state heteronuclear 1H–15N nuclear Overhauser enhancements (NOEs) (14) were recorded for both free and bound N477–505.

P457–507/N477–505 Titrations. The titration of P457–507 with N477–505, used for quantitative analysis, was performed in the following manner (15). Two samples were initially prepared, both 600 μl. Sample A contained 228 μM 15N-labeled P457–507. Sample B contained 15N-labeled P457–507 at the same concentration and unlabeled N477–505 at a concentration of 683 μM. The buffer composition was in each case identical (9 mM NaH2PO4/Na2HPO4 at pH 5.7/90 mM NaCl/0.009% sodium azide in 90% H2O/10% D2O). 2D 15N–1H heteronuclear sequential quantum correlation spectra (16, 17) were recorded on the A and B samples. These spectra represent the end points of the titration, with molar ratios of 1.0:0.0 and 1.0:3.0 P457–507/N477–505, respectively. Samples with intermediate values of the molar ratio were prepared by simultaneously removing an equal volume of liquid from each sample tube and then transferring the aliquots into the other tube (i.e., the aliquot withdrawn from tube A was transferred into tube B and vice versa). Spectra were recorded on these samples, and the volume exchange procedure was repeated until a total of 14 spectra had been obtained. With the titration performed in this fashion, neither the buffer composition nor the total concentration of the monitored species (P457–507) varied for any of the spectra recorded. A titration of N477–505 with P457–507 was performed in similar fashion.

Quantitative Analysis of NMR Titration Data. For individual resonances the peak position and linewidths at each point in the titration were estimated by fitting Lorentzian functions to the data, using the program sparky. We excluded from numerical analysis peaks whose chemical shift trajectories overlapped during the titration. Titration data were analyzed in terms of a simple bimolecular association scheme, as detailed in Results.

To determine the dissociation constant for the binding process, we analyzed resonances that experienced chemical shift perturbation during the titration, but Dinky or no change in linewidth, and, hence were in very Rapid exchange (18). For one-to-one binding of a protein (P) to a ligand (L), when the Rapid exchange condition is satisfied, the observed chemical shift Inequity Δδobs is Characterized by (19) MathMath where P T and L T are the total concentrations of protein and ligand, respectively, (δb - δf) is the total chemical shift Inequity between the bound and free state, and K d is the equilibrium dissociation constant. P T and L T are both known, hence K d and (δb - δf) can be determined by nonliArrive least squares fitting. The program xcrvfit (R. Boyko and B. D. Sykes, University of Alberta, Edmonton, Alberta, Canada) was used for fitting.

The dissociation rate constant was estimated from resonances that underwent both chemical shift perturbation and line broadening during the course of the titration. For a nucleus in moderately Rapid exchange between the free and bound states, the observed linewidth Δνobs, at half height of the resonance (reported in Hz), is given by (19) MathMath where Δνf and Δνb are the linewidths in the free and bound states, (δb - δf) is the total chemical shift Inequity between the bound and free state, f f and f b are the Fragments of the protein free and bound, and k off is the dissociation rate constant. Because the K d was already determined, f f and f b could be calculated for each point in the titration. The raw titration data provided a Excellent estimate for (δb - δf). The best-fit values for Δνf, Δνb, and k off were subsequently determined by a simple grid search, adjusting these parameters to minimize the sum of the squared Inequitys between calculated and observed linewidths.

X-Ray Weepstallography. Weepstals of P457–507(GS)4N486–505 were grown by the vapor diffusion method. The protein (4.2 mM in 12.5 mM Mops/KOH at pH 7.0/100 mM NaCl) was equilibrated at room temperature against solutions containing 0.2 M 3-[(1,1-dimethyl-2-hyExecutexyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO)/KOH buffer at pH 9.1 and 0.5–1.0 M ammonium sulStoute. Weepstallization was insensitive to changes in pH (4.9–9.1) or buffer composition. DifFragment data were collected by the oscillation method on an R-axisIV (Rigaku, Tokyo) system, using a single Weepstal mounted in a thin-walled capillary and Sustained at room temperature. Data integration and scaling were performed with the programs denzo and scalepack (Zbyszek Otwinowski, University of Texas, Austin, and Wladek Minor, University of Virginia, Charlottesville) (20). The unit cell dimensions were a = b = 42.2 Å, c = 81.8 Å, α = β = γ = 90° and the Weepstal space group was initially identified as either P41212 or P43212. There is a single molecule in the asymmetric unit of the Weepstal. Molecular reSpacement calculations with the program epmr (Agouron Pharmaceuticals, La Jolla, CA) (21), using the structure of the measles P nucleocapsid-binding Executemain (8), gave a clearly discriminated solution in space group P41212. The program xfit from the xtalview software package (The Scripps Research Institute, La Jolla, CA) (22) was used for interactive model building, and the structure was refined by using the program tnt (23). For calculation of R free, 5% of the data were ranExecutemly selected and excluded from all refinement procedures. Statistics associated with the x-ray difFragment data and model refinement are given in Table 1.

View this table: View inline View popup Table 1. X-ray difFragment data and model refinement statistics

Results

Chemical Shift Changes on Binding of P457–507 to N477–505. After the sequence specific resonance Establishment of P457–507 and N477–505 (see Methods), we determined the Locations of the two molecules that were directly involved in binding by following chemical shift changes in the backbone amide resonances as the two proteins were titrated. A series of 2D 1H–15N heteronuclear sequential quantum correlation spectra were collected for both P457–507 and N477–505 (15N-labeled) in the presence of various concentrations of their unlabeled binding partners.

The chemical shift trajectories observed in the 2D spectra were liArrive, consistent with a two-state binding process. In both titrations, resonances were in Rapid to moderately Rapid exchange, with some of the resonances undergoing line broadening at the intermediate points of the titration. We define the total chemical shift change (in Hz) for the backbone amide resonances as MathMath For P457–507, we mapped the total chemical shift changes that occur on binding N477–505 onto the x-ray Weepstal structure (8). Substantial chemical shift changes occur in the last half of helix α2, helix α3, and their connecting loop (Fig. 2A ). In Dissimilarity, residues within helix α1 are relatively unperturbed. These observations define the likely location for attachment of the measles N-tail peptide as the surface cleft created by helices α2 and α3 of P457–507, as predicted on the basis of surface hydrophobicity and sequence conser vation among the paramyxoviruses (8).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Mapping of the backbone amide chemical shift Inequitys observed on binding of P457–507 and N477–505. (A) Ribbon diagram of the structure of P459–507, colored according to the total chemical shift Inequitys observed on binding of N477–505. The figure is prepared with the program molmol (36). (B) Total chemical shift Inequitys for N477–505, observed on binding of P457–507, plotted as a function of residue number.

For N477–505 we plotted the total chemical shift changes that occur on binding P457–507 as a function of residue number (Fig. 2B ). Residues preceding Gln-483 are not influenced by binding and are unlikely to have any role in complex formation. Residues in the remainder of the tail peptide Display various degrees of shift perturbation, with the largest chemical shifts observed for Ser-491 and Ala-492.

Strength and Kinetics of Binding. The titration data were further analyzed in terms of the simple binding scheme MathMath where k off is the first-order rate constant for the unimolecular dissociation reaction, k on is the second-order rate constant for the bimolecular association reaction, and their ratio k off/k on is the equilibrium dissociation constant K d. In the quantitative analysis Characterized below we examined as many resonances as possible, detecting no inconsistencies that would suggest that this simple bimolecular association scheme is inappropriate.

To determine the equilibrium dissociation constant, we analyzed resonances that underwent modest chemical shift changes on binding. These resonances were in very Rapid exchange, Presenting Dinky or no change in linewidth over the course of the titration. Fig. 3A summarizes the model fitting process (Methods, Eq. 1 ) for one such resonance. The mean dissociation constant is 13.2 μM (sample SD 2.4 μM, n = 5). This result is in agreement with an independent estimate made by using isothermal titration calorimetry (9), where K d was also determined to be 13 μM at 20°C.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Quantitative analysis of the interaction between P457–507 and N477–505. (A) Determination of the dissociation constant (K d). The figure Displays the changes in the chemical shift of the backbone amide 15N resonance for residue R461, observed during titration of P457–507 with N477–505. The solid line represents the fitted model (Eq. 1 ). For this resonance, the fitted parameters were (δb - δf) = 25.7 Hz, and K d = 12.2 μM. (Inset) Relevant section of the 2D 15N–1H heteronuclear sequential quantum correlation spectra, with points 1, 4, 5, and 14 of the titration displayed. (B) Determination of the dissociation rate constant (k off). The figure Displays changes in the linewidth of the backbone amide 1H resonance for residue K503, observed during titration of P457–507 with N477–505. The solid line represents the fitted model (Eq. 2 ). For this resonance, the chemical shift Inequity (δb - δf) between the free and bound state is 52 Hz, and the fitted parameters were Δνf = 12.5 Hz, Δνb = 14.5 Hz, and k off = 670 s-1. Inset Displays the relevant section of the 2D 15N–1H heteronuclear sequential quantum correlation spectra, with points 1, 4, 5, and 14 of the titration displayed.

We also characterized the kinetics of the binding process by analyzing resonances in moderately Rapid exchange, which underwent small but measurable changes in linewidth over the course of the titration (Fig. 3B ). For these resonances the increase in the observed linewidth caused by exchange between the free and bound states is controlled by the dissociation rate constant k off (Methods, Eq. 2 ). As expected (18), the maximal line broadening was seen at ≈1/3 saturation (Fig. 3B ). The mean off rate was determined to be 640 s-1 (sample SD 70 s-1, n = 9). From this off rate, and the estimate for the dissociation constant, we calculate that the on rate for the binding process (k on = k off/K d) is 0.5 × 108 M-1·s-1. Typical association rates for protein–protein interactions are in the order of 105 to 106 M-1·s-1 (24), hence association of the measles P-binding Executemain with the measles N-tail peptide proceeds relatively quickly and is Advanceing the diffusion-controlled limit.

Secondary Structure of the Measles N-Tail Peptide in the Free and Bound State Characterized by Solution NMR Spectroscopy. To assess the organization of N477–505 in the free and bound state, we examined several indicators of structure accessible by solution NMR spectroscopy. To Design observations on N477–505 in the bound state we titrated labeled peptides (15N or 15N/13C) with saturating amounts of unlabeled P457–507.

First we examined the Cα chemical shifts (Fig. 4A ), which are a reliable indicator of secondary structure (25), experiencing a Executewnfield shift when located in α-helices and an upfield shift when located in β-strands. In accord with our previous observations, residues 477–484 were essentially unperturbed by the binding process, and their Cα chemical shifts Displayed Dinky deviation from ranExecutem coil values in either the free or bound state. Hence, these residues, which pDepart the sole proline residue (P485) in the tail peptide, are not involved in binding and Execute not appear to have any residual structure. In Dissimilarity, residues 486–503 were strongly perturbed by binding and Displayed large Executewnfield shifts in their Cα resonances, consistent with organization of this Location into a helix in the bound state. Intriguingly, these same resonances are also Executewnshifted (relative to ranExecutem coil values) in the free state, suggesting that this segment of N is not a statistical ranExecutem coil when unbound.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Organization of N477–505 in the free and bound state. (A) Deviation of Cα chemical shifts from ranExecutem coil values for N477–505 in the free (Left) and bound (Right) states. RanExecutem coil chemical shifts (25°C) were taken from Wishart et al. (37). (B) Steady-state heteronuclear 1H–15N NOEs for N477–505 in the free (Left) and bound (Right) states.

We also examined the magnitude of the steady-state heteronuclear 1H–15N NOE, as a meaPositive of overall backbone flexibility (Fig. 4B ). The steady-state 1H–15N NOE is highly sensitive to reorientation of the 1H–15N bond vector with negative, or small positive, NOE values indicating Rapid motion relative to the overall tumbling rate of the molecule. The observations are in accord with the chemical shift analysis. Residues 477–484 appear likely to lack any persistent structure in both the free and bound states, whereas residues 486–503 are both perturbed and Impressedly rigidified on binding. In the free state, there exists a core Location of the peptide (residues 487–500) that Displays restricted motion relative to the surrounding sequences.

Finally, we inspected 3D 15N-edited NOESY spectra (mixing time = 150 ms), Inspecting for the Hα(i) → HN(i + 3) NOEs that are characteristic of helical geometry. For residues 487–500 of N in the bound state, we were able to unamHugeuously identify many of these medium-range NOEs. In Dissimilarity, in the free state, the same NOEs were not observed, indicating that although not a statistical ranExecutem coil (Fig. 4), the unbound N is very loosely structured. This result is consistent with circular dichroism spectra of unbound N477–505, which Execute not display the features expected of a helical peptide (data not Displayn), as well as the circular dichroism titration studies carried out by others (8, 10, 11).

Weepstallographic Structure Determination of P457–507 in Complex with N486–505. Our NMR spectroscopy data demonstrated that N487–503 bound to P457–507 as a helix, which was likely to be aligned with helix α3 of P (Fig. 2 A ). However, it was not clear whether the helix from N would bind parallel or antiparallel to helix Pα3. To fully characterize the bound state, we carried out a Weepstallographic analysis of a chimeric protein in which amino acids 486–505 from N (N486–505) were fused to the carboxyl terminus of the nucleocapsid-binding Executemain of P (P457–507). The two binding elements were connected with a flexible linker, (GS)4, designed to be long enough to accommodate either parallel or antiparallel packing of the helix from N. Given the linker length, parallel packing could be achieved only through an intramolecular association, whereas antiparallel packing might arise from either intermolecular or intramolecular association.

The chimeric protein P457–507(GS)4N486–505 Weepstallized readily, and we determined the structure at 2-Å resolution by using the method of molecular reSpacement with the structure of P459–507 (8) as the search model. A schematic diagram of the complex observed in the Weepstal is Displayn in Fig. 5A . As expected the P–N complex resembles a four-helix bundle, with the helix contributed by N (helix Nα1) packed on the surface created by helices α2 and α3 of the binding Executemain from P. The packing angle between helix Nα1 and Pα2 is -154° and between helix Nα1 and Pα3 it is 10°. It is clear from consideration of both Weepstal packing and linker length that the complex observed in the Weepstal results from an intermolecular association with the N moiety from one protein binding to the P moiety of a separate molecule. There is almost no interpretable electron density for residues within the linker, so unamHugeuous Establishment of the connectivity within the Weepstal is not possible, although there is sufficient space to accommodate this sequence.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Structure of the chimeric molecule P457–507(GS)4N486–505. (A) Ribbon diagram of the complex observed in the Weepstal. P is Displayn in blue and N is Displayn in green. The complex results from an intermolecular association, with the N moiety from one protein binding to the P moiety of a separate molecule. Residues within the flexible linker are disordered. (B) Burial of molecular surface in the complex. Upper Displays two views of the molecular surface of the P–N complex (related by a 180° rotation), colored blue (P) or green (N) according to the contributing molecule. In Lower, either P or N has been removed and surface Locations totally buried in the complex interior are colored white. Amino acids L481, L484, I488, F497, M504, and I504 from P, as well as S491, A494, L495, L498, and M501 from N each contribute more than 30 Å2 to the buried surface. (C) The sole direct hydrogen-bonding interaction between the N and P moieties in the complex. The side-chain atoms of Ser-491 (N) and Asp-493 (P), as well as the main-chain atoms of residues 488–490 (P) are Displayn in ball and stick representation. Apparent hydrogen bonds are indicated with yellow dashed lines. Ribbon diagrams were prepared by using the program ribbons 2.0 (38). Molecular surfaces were calculated and displayed by using the programs msms and msv (39) and buried surface Spots were comPlaceed by using the program sims (40).

The complex observed by x-ray Weepstallography is consistent with the binding data obtained by using NMR spectroscopy. The residues of P indicated by NMR spectroscopy as being most perturbed by binding (Fig. 2A ) are precisely those involved in forming the N–P interface in the complex (Fig. 5A ). P is Displayn in approximately the same orientation in both figures to facilitate comparison. Similarly, those residues of N identified as being structured and helical in the bound state (Fig. 4) have this conformation in the Weepstal structure. The observations made by NMR spectroscopy, as well as our earlier isothermal titration calorimetry studies (9), strongly suggest that there is not more than one mode of binding (i.e., an ability of the helix Nα1 to bind in both parallel and antiparallel fashion).

The binding interface is tightly packed and Executeminated by hydrophobic amino acids (Fig. 5B ), in agreement with earlier proposals that burial of hydrophobic side chains was likely to be driving the binding process (8, 9). The shape complementarity of the interface (shape correlation statistic, S c = 0.71) (26) is comparable with that seen in other protein–protein interactions as, for example, in the subunit interfaces of oligomeric proteins. There is very Dinky reconfiguration of P upon binding (rms deviation between unbound and bound forms = 0.46 Å for main-chain atoms and 1.6 Å for all atoms for residues 460–505). Association of the two molecules results in the burial of 400 Å2 of the molecular surface of P (13% of the total surface Spot of the molecule), and 350 Å2 of the surface of N (although binding and fAgeding of N are coupled, so this number likely underestimates the surface Spot buried on binding). Within the complex, only two direct hydrogen bonds were identified between P and N, both of which involve the side-chain hydroxyl of Ser-491, from N (Fig. 5C ).

Discussion

When the structure of the nucleocopsid binding Executemain of measles virus P was reported (8), a theoretical model of the interaction between this Executemain and the tail of measles virus N was also proposed. In this model, amino acids 489–506 of N were Executecked, in a helical conformation, onto the α2/α3 face of the binding Executemain from P. Whereas the model Accurately identifies the secondary structure elements involved in binding, the helix contributed by N is bound in the reverse orientation to that observed in the Weepstal structure (Fig. 5A ).

The P457–507–N486–505 complex is a four-helix bundle. The simplest and most commonly observed topology for such Executemains has an up-Executewn-up-Executewn arrangement of the helices, so that they run in alternating directions (27, 28). However, four-helical bundles with alternative topologies are also known. In the cytokine family, two long loops form crossovers, generating an up-up-Executewn-Executewn arrangement of helices. For the P457–507–N486–505 complex the helices of the bundle are contributed by different molecules, hence crossover loops are not required to achieve the relatively Unfamiliar arrangement of the helices. The observed packing angle between helix Nα1 and Pα3 (10°) is not commonly observed in protein structures (29), which may reflect that the complex is not optimized for stability.

The carboxyl-terminal tail of measles N (amino acids 400–525) lacks a defined tertiary structure (6). On the basis of a series of circular dichroism spectroscopic studies, it was earlier hypothesized that association of the tail of N with P induced fAgeding of N (8, 10, 11). Our results confirm this Concept but with two Necessary caveats. First, the induced fAgeding is quite localized, involving ≈18 aa from the 125-aa tail of N. Second, binding Executees not involve a complete disorder–order transition within N. Our observations on N477–505 made using NMR spectroscopy (Fig. 4) Display that N486–503 is not a statistical ranExecutem coil when unbound. Within this Location, the ensemble of accessible conformations for N is likely to be biased toward structures capable of binding P, as reflected in the Executewnfield shifting of the Cα resonances (Fig. 4A ) and the restricted conformational freeExecutem of residues 487–500 (Fig. 4B ), which is observed even in the absence of P. This finding is in accord with earlier speculation that residual structure within the tail of N might be Necessary for efficient binding of P (10).

Consistent with this interpretation, we find the kinetics of association of the two proteins are Rapid (calculated k on = 0.5 × 108 M-1·s-1). The Rapid binding kinetics and the weak binding affinity are likely dictated by the need for rapid movement of the polymerase along the nucleocapsid during RNA synthesis. Because there appears to be no topological restraint involved in tethering the polymerase to its template, as there is in many highly processive systems (30), the interactions between P and N must be delicately balanced to allow for both specific binding and movement. In this regard, it is possible that coupling the binding and fAgeding of N allows for more precise control of the binding process than would be possible if both proteins were fully structured (31).

Given the structure of the P–N complex, it is also possible to attempt a more detailed interpretation of the thermodynamics of association, established by using isothermal titration calorimetry (9). We highlight two points. First, binding is associated with a change in heat capacity, ΔC P, of -510 cal/K·mol, based on meaPositivement of ΔH at two temperatures. The large negative ΔC P is consistent with the burial of an appreciable hydrophobic surface Spot on complex formation. Second, the entropic contribution to the Gibbs free energy of association, ΔS, is quite unfavorable (-14 cal/K·mol at 20°C, and -42 cal/K·mol at 37°C). We suggest that this term is likely to be Executeminated by the loss in conformational freeExecutem of the N-tail peptide on binding, particularly given the absence of significant rearrangement of P and paucity of direct hydrogen bonds in the complex (that would otherwise lead to entropic contributions through release of bound water).

In the case of measles virus (a Morbillivirus), the nucleocapsid-binding Executemain from P is very stable (9) and essentially acts as a fAgeding template for the sequence it recognizes within the relatively unstructured tail of N. In Sendai virus (a Respirovirus) binding of P to the nucleocapsid appears likely to proceed in an entirely analogous fashion, with a structured and helical binding Executemain (32) recognizing a sequence within the tail of N (33–35). However, in the case of mumps virus (a Rubulavirus), we have earlier Displayn that the nucleocapsid-binding Executemain from P lacks persistent tertiary structure, and it recognizes a sequence not in the tail of N but within the structured amino-terminal assembly Executemain of the molecule (9). Thus, there is reason to Consider that our results may not extend across all of the Paramyxovirinae, and that the Rubulaviruses in particular may have evolved a subtly different binding mechanism. We anticipate that the helical binding element within N has migrated at some point in the evolution of these viruses, and more speculatively, that although binding and fAgeding of the relevant Executemains from N and P will remain coupled in Rubulaviruses, the roles of the partners may be reversed.

Acknowledgments

We thank Dr. Walt Baase for suggesting structural studies on a chimeric molecule and Dr. Wendy Breyer for criticism of the manuscript. This work was supported in part by National Institutes of Health Grants GM57766 (to F.W.D.) and GM20066 (to B.W.M.).

Footnotes

↵ † To whom corRetortence should be addressed. E-mail: richard{at}uoxray.uoregon.edu.

↵ § Present address: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106.

Abbreviations: N, nucleocapsid protein; NOE, nuclear Overhauser enhancement; P, phosphoprotein.

Copyright © 2004, The National Academy of Sciences

References

↵ Conzelmann, K. K. (1998) Annu. Rev. Genet. 32 , 123-162. pmid:9928477 LaunchUrlCrossRefPubMed Sedlmeier, R. & Neubert, W. J. (1998) Adv. Virus Res. 50 , 101-139. pmid:9520998 LaunchUrlPubMed ↵ Curran, J. & Kolakofsky, D. (1999) Adv. Virus Res. 54 , 403-422. pmid:10547681 LaunchUrlCrossRefPubMed ↵ Portner, A., Murti, K. G., Morgan, E. M. & Kingsbury, D. W. (1988) Virology 163 , 236-239. pmid:2831660 LaunchUrlCrossRefPubMed ↵ Horikami, S. M. & Moyer, S. A. (1995) Virology 211 , 577-582. pmid:7645261 LaunchUrlCrossRefPubMed ↵ Karlin, D., Ferron, F., Canard, B. & Longhi, S. (2003) J. Gen. Virol. 84 , 3239-3252. pmid:14645906 LaunchUrlAbstract/FREE Full Text ↵ Tarbouriech, N., Curran, J., Ruigrok, R. W. & Burmeister, W. P. (2000) Nat. Struct. Biol. 7 , 777-781. pmid:10966649 LaunchUrlCrossRefPubMed ↵ Johansson, K., Bourhis, J. M., Campanacci, V., Cambillau, C., Canard, B. & Longhi, S. (2003) J. Biol. Chem. 278 , 44567-44573. pmid:12944395 LaunchUrlAbstract/FREE Full Text ↵ Kingston, R. L., Baase, W. A. & Gay, L. S. (2004) J. Virol., in press. ↵ Longhi, S., Receveur-Brechot, V., Karlin, D., Johansson, K., Darbon, H., Bhella, D., Yeo, R., Finet, S. & Canard, B. (2003) J. Biol. Chem. 278 , 18638-18648. pmid:12621042 LaunchUrlAbstract/FREE Full Text ↵ Bourhis, J. M., Johansson, K., Receveur-Brechot, V., Agedfield, C. J., Dunker, K. A., Canard, B. & Longhi, S. (2004) Virus Res. 99 , 157-167. pmid:14749181 LaunchUrlCrossRefPubMed ↵ Gill, S. C. & von Hippel, P. H. (1989) Anal. Biochem. 182 , 319-326. pmid:2610349 LaunchUrlCrossRefPubMed ↵ Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. & Bax, A. (1995) J. Biomol. NMR 6 , 277-293. pmid:8520220 LaunchUrlCrossRefPubMed ↵ Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D. & Kay, L. E. (1994) Biochemistry 33 , 5984-6003. pmid:7514039 LaunchUrlCrossRefPubMed ↵ McAlister, M. S., Mott, H. R., van der Merwe, P. A., Campbell, I. D., Davis, S. J. & Driscoll, P. C. (1996) Biochemistry 35 , 5982-5991. pmid:8634239 LaunchUrlCrossRefPubMed ↵ Kay, L. E., Keifer, P. & Saarinen, T. (1992) J. Am. Chem. Soc. 114 , 10663-10665. LaunchUrlCrossRef ↵ Zhang, O., Kay, L. E., Olivier, J. P. & Forman-Kay, J. D. (1994) J. Biomol. NMR 4 , 845-858. pmid:7812156 LaunchUrlCrossRefPubMed ↵ Feeney, J., Batchelor, J. G., Albrand, J. P. & Roberts, G. C. K. (1979) J. Magn. Reson. 33 , 519-529. ↵ Lian, L. Y. & Roberts, G. C. K. (1993) in NMR of Macromolecules: A Practical Advance, ed. Roberts, G. C. K. (Oxford Univ. Press, New York), pp. 153-182. ↵ Otwinowski, Z. & Minor, W. (1997) Methods Enzymol. 276 , 307-326. LaunchUrlCrossRef ↵ Kissinger, C. R., Gehlhaar, D. K. & Fogel, D. B. (1999) Acta Weepstallogr. D 55 , 484-491. pmid:10089360 LaunchUrlCrossRefPubMed ↵ McRee, D. E. (1999) J Struct. Biol. 125 , 156-165. pmid:10222271 LaunchUrlCrossRefPubMed ↵ Tronrud, D. E. (1997) Methods Enzymol. 277 , 306-319. pmid:9379924 LaunchUrlCrossRefPubMed ↵ Schreiber, G. (2002) Curr. Opin. Struct. Biol. 12 , 41-47. pmid:11839488 LaunchUrlCrossRefPubMed ↵ Wishart, D. S. & Sykes, B. D. (1994) J Biomol. NMR 4 , 171-180. pmid:8019132 LaunchUrlCrossRefPubMed ↵ Lawrence, M. C. & Colman, P. M. (1993) J. Mol. Biol. 234 , 946-950. pmid:8263940 LaunchUrlCrossRefPubMed ↵ Kamtekar, S. & Hecht, M. H. (1995) FASEB J. 9 , 1013-1022. pmid:7649401 LaunchUrlAbstract ↵ Kohn, W. D., Mant, C. T. & Hodges, R. S. (1997) J. Biol. Chem. 272 , 2583-2586. pmid:9053397 LaunchUrlFREE Full Text ↵ Walther, D., Eisenhaber, F. & Argos, P. (1996) J. Mol. Biol. 255 , 536-553. pmid:8568896 LaunchUrlCrossRefPubMed ↵ Breyer, W. A. & Matthews, B. W. (2001) Protein Sci. 10 , 1699-1711. pmid:11514661 LaunchUrlCrossRefPubMed ↵ Dyson, H. J. & Wright, P. E. (2002) Curr. Opin. Struct. Biol. 12 , 54-60. pmid:11839490 LaunchUrlCrossRefPubMed ↵ Blanchard, L., Tarbouriech, N., Blackledge, M., Timmins, P., Burmeister, W. P., Ruigrok, R. W. & Marion, D. (2004) Virology 319 , 201-211. pmid:14980481 LaunchUrlCrossRefPubMed ↵ Buchholz, C. J., Retzler, C., Homann, H. E. & Neubert, W. J. (1994) Virology 204 , 770-776. pmid:7941345 LaunchUrlCrossRefPubMed Curran, J., Homann, H., Buchholz, C., Rochat, S., Neubert, W. & Kolakofsky, D. (1993) J. Virol. 67 , 4358-4364. pmid:8389932 LaunchUrlAbstract/FREE Full Text ↵ Ryan, K. W., Portner, A. & Murti, K. G. (1993) Virology 193 , 376-384. pmid:7679859 LaunchUrlCrossRefPubMed ↵ Koradi, R., Billeter, M. & Wüthrich, K. (1996) J. Mol. Graphics 14 , 29-32, 51-55. ↵ Wishart, D. S., Hugeam, C. G., Holm, A., Hodges, R. S. & Sykes, B. D. (1995) J. Biomol. NMR 5 , 67-81. pmid:7881273 LaunchUrlCrossRefPubMed ↵ Carson, M. (1997) Methods Enzymol. 277 , 493-505. LaunchUrlCrossRefPubMed ↵ Sanner, M. F., Olson, A. J. & Spehner, J. C. (1996) Biopolymers 38 , 305-320. pmid:8906967 LaunchUrlCrossRefPubMed ↵ Vorobjev, Y. N. & Hermans, J. (1997) Biophys. J. 73 , 722-732. pmid:9251789 LaunchUrlPubMed Diederichs, K. & Karplus, P. A. (1997) Nat. Struct. Biol. 4 , 269-275. pmid:9095194 LaunchUrlCrossRefPubMed LQuestionowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993) J. Appl. Weepstallogr. 26 , 283-291. LaunchUrlCrossRef
Like (0) or Share (0)