Melinda Roy1, Leslie LJose N. Onuchic2 and
1Departments of Chemistry andBiochemistry, University ofCalifornia at San Diego, La JollaCA 92093-0359,USA
2Center for TheoreticalBiological Physics, University ofCalifornia at San Diego, La JollaCA 92093-0374, USA
doi:10.1016/j.jmb.2005.02.059 J. Mol. Biol. (2005) 348, 335347shift between native and 0.8 M Gdn-HCl, which were determinedexperimentally. This correlation demonstrates that structural changeswithin the native ensemble of IL-1b are, at least partially, governed bythe principle of minimal energetic frustration.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: interleukin-1b; b-trefoil; topology; molecular dynamics; Go-model*Corresponding author0022-2836/$ - see front matter q 2005 E
Present address: D. K. Heidary, VAbbreviations used: IL-1b, interle
correlation; RMSD, root-mean-squarisomerase; MD, molecular dynamic
E-mail address of the corresponde Population Ensemble throughlogy
. Chavez2, John M. Finke2, David K. Heidary1
Patricia A. Jennings1*
A minimalist Go-model, with no energetic frustration in the nativeconformation, has been shown to describe accurately the folding pathwayof the b-trefoil protein, interleukin-1b (IL-1b). While it appears that thesemodels successfully model transition states and intermediates between theunfolded and native ensembles, it is unclear how accurately they capturesmaller, yet biologically relevant, structural changes within the nativeensemble after energetic perturbation. Here, we address the followingquestions. Can a simple Go-model of interleukin-1b, based on nativetopology, describe changes in structural properties of the native ensembleas the protein stability is changed? Or is it necessary to include a moreexplicit representation of atoms, electrostatic, hydrogen bonding, and vander Waals forces to describe these changes?
The native ensemble of IL-1b was characterized using a variety ofexperimental probes under native (0 M NaCl, guanidine hydrochloride(Gdn-HCl)), moderately destabilized (0 M NaCl, 0.8 M Gdn-HCl), and inmoderate salt concentration (0.8 M NaCl, 0 M Gdn-HCl). Heteronuclear1H15N nuclear Overhauser effect spectroscopy (NOESY) and hetero-nuclear single quantum correlation (HSQC) NMR spectra confirmed thatthe b-trefoil global fold was largely intact under these three conditions.However, 25 of the 153 residues throughout the chain did demonstrate 13Cand 1H15N chemical shifts when perturbed with 0.8 M NaCl or Gdn-HCl.Despite large differences in protection factors from solvent hydrogen-deuterium exchange for all residues between stable (0 M Gdn-HCl) anddestabilized (0.8 M Gdn-HCl) IL-1b, no difference in steady-state 15N1HNOE enhancements were measured. Thus, the chemical shifts correlatewith a global but limited increase in residue flexibility in the presence ofGdn-HCl. Minimalist simulations highlight the regions of greatest positionThe Native Energy Landscape for Interleukin-1b.Modulation of thlsevier Ltd. All rights reserved.
ertex Pharmaceuticals Inc., San Diego, CA 92121,USA.ukin-1b; Gdn-HCl, guanidine hydrochloride; HSQC, heteronuclear single quantume deviation; NOESY, nuclear Overhauser effect spectroscopy; TIM, triose phosphates; pdb, Protein Data Bank.ing author: email@example.com
circular dichroism signal remains largely16,26,27,32,35,36
the case of IL-1b, it remains unclear whether thehyperfluorescent ensemble populated at 0.8 MIntroduction
Simple theoretical models demonstrate that thefolding pathways of proteins with funneled energylandscapes are nearly completely determined bythe energetic requirements of adopting the finalnative state topology,18 with a few notable excep-tions in simulations of the folding of symmetricproteins.9 These models exploit topological infor-mation contained in the X-ray crystal structuresand are able to capture the dominant structuraland thermodynamic properties of the foldingmechanisms of both small proteins and morecomplex proteins such as interleukin-1b (IL-1b)and dihydrofolate reductase.4,10 These studies areespecially noteworthy, as both proteins are of asimilar size yet fold through different intermediatestructures.11,12 Thus, topological features of thenative protein are sufficient to predict the generalstructures and thermodynamic properties of thestates populated between distinct unfolded andfolded protein ensembles. Nonetheless, it remainsto be seen whether more subtle structural andthermodynamic changes, which occur during per-turbation of a single ensemble, i.e. the native stateensemble, can also be captured with these simplemodels. A recent study has shown that localperturbations induced by loop inserts can besimulated in a simple theoretical Go-model ofchymotrypsin inhibitor 2 (CI2) nearly exactly asobserved in experiments.13 Here, we addresswhether this agreement will hold for globalperturbation of the native IL-1b ensemble bychemical denaturants.
The inflammatory cytokine IL-1b has diverse andcomplex actions throughout the body and thebrain.14 IL-1b is partially unfolded during secretionfrom activated macrophages and monocytes,15 andis unfolded during in vitro aggregation16 and in vivoprecipitation in the brain during amyloid plaqueevolution in Alzheimers disease.17,18 Thus, theconformational dynamics that control the popu-lation of unfolded or partially unfolded stateswithin the native state ensemble are of growinginterest. The kinetic unfolding mechanism of IL-1bunder strongly denaturing conditions reveals apartially rugged landscape in which unfoldingseems to be best represented by the kineticprogression towards an ensemble of lessstructurally organized structures, rather thandiscrete unfolding intermediates.19
IL-1b displays the archetypal b-trefoil globalfold,20,21 where six b-strands form a b-barrel(b1, b4, b5, b8, b9, and b12) and six b-strandsform a three-hairpin cap (b2Cb3, b6Cb7, andb10Cb11).20,22,23 A discrete folding intermediatepopulates during the kinetic refolding of IL-1b,11,24,25
the structure of which has been captured withminimalist theoretical models.4 The equilibriumunfolding transition as monitored by CD spec-
336troscopy,26,27 Trp120 fluorescence average wave-length and anisotropy,28 and DSC29 is well fit by atwo-state unfolding model, which implies noGdn-HCl is comprised of partially unfolded inter-mediates or slightly perturbed members of thenative ensemble. The present study investigates thestructural properties of the IL-1b hyperfluorescentstate populated at 0.8 M Gdn-HCl, revealing thestructural details of this ensemble. This study alsoemploys molecular dynamics simulations to testwhether a minimalist Ca-only protein model with afunneled energy landscape is sufficient to capturethe measured properties of the native ensemble.
Experimental measurements of the native basin
In IL-1b, the fluorescence intensity increases in alinear fashion with increasing concentrations ofNaCl, up to a maximum and constant value at andabove 3 M NaCl. In contrast, the equilibriumdenaturation profile of IL-1b exhibits an initialincrease in fluorescence signal that plateaus at 0.8 MGdn-HCl and then subsequently decreases between1 M and 2 M Gdn-HCl. The initial increase influorescence represents a hyperfluorescent ensem-ble populated at 0.8 M Gdn-HCl and is similar tothat populated at high NaCl concentration (data notshown). The decreasing signal between 1 M and2 M Gdn-HCl is coincident with the transition thatmonitors the cooperative unfolding of the protein asmeasured with a variety of probes.16,26,27,35,36 Con-trol experiments demonstrate that isolated tyrosineand tryptophan residue fluorescence increases withunchanged. Recent studies have usedH/2H exchange to probe proteins at low levels ofdenaturant, which destabilized the native ensemblebut did not produce a hyperfluorescent state.33,34 Instable intermediates. In contrast, IL-1b exhibitshyperfluorescence, maximal near 0.8 M guanidinehydrochloride (Gdn-HCl), during the equilibriumGdn-HCl unfolding transition monitored withintrinsic tryptophan fluorescence and the transitioncannot be fit to either a two-state or a three-statefit.28 This hyperfluorescence can be explained byeither population of a metastable equilibriumintermediate and/or structural transitions withina conformationally flexible native state ensemble.30
Although not hyperfluorescent, another b-trefoilprotein, fibroblast growth factor 1 (FGF-1), alsopossesses a highly native-like intermediatepopulated during equilibrium Gdn-HClunfolding.31
In equilibrium titrations with a chemicaldenaturant, IL-1b exhibits hyperflourescence atlow denaturant concentrations, although the
Native Energy Landscape for IL-1bNaCl and Gdn-HCl concentration but can accountfor only half of the hyperfluorescence observed inIL-1b at the same NaCl and Gdn-HCl concentration.
1H15N HSQC chemical shifts
The 1H15N HSQC spectra of native IL-1b(yellow) and IL-1b in 0.8 M Gdn-HCl (red) and in0.8 M NaCl (blue) are superimposed in Figure 1.Both Gdn-HCl and NaCl cause small changes in thechemical shifts of many signals, but the overallpattern and chemical shift dispersion of backboneamide proton and nitrogen resonances remainsimilar, indicating a largely NMR averaged native-like global fold under all these conditions. Inaddition, the 3D HSQC-NOESY spectra (data notshown) of native IL-1b or IL-1b in 0.8 M Gdn-HCl orNaCl show no significant changes and no changesin tertiary structure, except for additional NOEsignals associated with Glu111. Relative to thenative state resonances, most of the resonancesthat shift in 0.8 M Gdn-HCl are similar to those thatshift in 0.8 M NaCl. However, there are residueswhere the effect of denaturant is distinct from theeffect of salt and these residues are highlighted inFigure 1 (residues 4, 24, 51, 76, 83, 92, 96, 108, 142and 153). A few of the resonance signals disappear(e.g. Phe112, Ile143) and are likely the result of
linewidth broadening due to intermediate exchangebetween alternate conformations.
Ca chemical shifts
The deviations of the 13Ca chemical shifts fromrandom coil values are dependent on the f,4backbone dihedral angles and commonly used tocharacterize the secondary structure of folded andunfolded proteins at the level of individualresidues.37 While 1H15N HSQC chemical shiftsare highly sensitive to both structural changes andsolution conditions (salt, pH), 13Ca carbon chemicalshifts are largely indicative of changes in backbonedihedrals and are not as sensitive to solutionconditions. An observed 13Ca chemical shiftindicates a preferential population of either the aor b-region of f,4 space for a given residue anddeviations in this shift upon Gdn-HCl destabiliza-tion can be interpreted as a change in the averageconformation of that dihedral.
For IL-1b, relative 13Ca chemical shift deviationswere derived from HNCA spectra (see Materials
Figure 1. Two-dimensional1H15N HSQC spectra of IL-1b in
Native Energy Landscape for IL-1b 3370 M NaCl/Gdn-HCl (yellowpeaks), 0.8 M NaCl (blue peaks),and 0.8 M Gdn-HCl (red peaks).
Peaks of residues with the mostsignificant shifts in NaCl andGdn-HCl are labeled.
and Methods). Residue-specific 13Ca ppm devi-ations relative to native IL-1b in the presence of0.8 M Gdn-HCl (red) and 0.8 M NaCl (blue) areshown in Figure 2. If the native structure remainsunchanged in Gdn-HCl and NaCl, relative chemicalshift deviations will be near zero. Residues willexhibit a relative deviation near 0 when native shiftsare unaffected while residues near 1 when shifts aresimilar to random coil. Relative deviations greaterthan 1 and less than 0 result from chemical shiftsaway from both native and random coil (see Figure2). Residues with the largest 13Ca shifts in 0.8 MGdn-HCl are 14, 22, 42, 48, 52, 91, 99, 103, 120, 123,132, 135, 136, 138, and 140.
Minimal variation in the steady-state 1H15NNOE values was observed upon addition of lowlevels of denaturant or salt, except for a decrease atGlu111 (data not shown), consistent with theobservation of the interaction of this side-chainwith a guanidinium ion. These results indicate thatthe fast motions of the backbone residues detectedwith the steady-state 1H15N NOE experiment donot vary between native conditions, 0.8 M Gdn-
native IL-1b are observed due to their stable
of the b-barrel and hairpin cap.38 In order to probethe strength and stability of the IL-1b hydrogen-bond network in the presence of low levels of saltand denaturant, we measured residue-specific HN
solvent exchange protection factors for native IL-1b,and IL-1b in the presence of 0.8 M Gdn-HCl and0.8 M NaCl. In Figure 3 the 73 HN protection factorsare plotted by residue number. Protection of theamide hydrogen bonds from solvent exchange innative IL-1b is observed within the 12 b-strands ofthe barrel and cap, and the small 310 helix (yellow).The protection factors in low levels of salt (blue) areonly moderately perturbed with a slight destabili-zation of the b-sheet hydrogen-bond network.However, in denaturant (red), there is a severeglobal perturbation of the entire H-bond networkfrom 106 to 103. Although no residues retain nativesolvent H/2H exchange protection, there are sevenHN groups with moderate protection factors thatexhibit slightly higher protection factors (104) thanthe surrounding protons in the presence of Gdn-HCl (Gln15, Leu26, Ile56, Leu80, Ile106, Asn129,Gln149).
Modeling the native basin with moleculardynamics simulations
338 Native Energy Landscape for IL-1bformation of hydrogen bonds between the b-strandsHCl, and 0.8 M NaCl.
Amide HN solvent exchange protection factors
The 73 slowly exchanging amide protons inFigure 2. Relative D d13C for13Ca chemical shifts of IL-1b r
between native and 0.8 M Gdn-HCl (red points). Residues windicated.A proteins native basin comprises an ensembleof states, which are predominantly folded globallybut may be unfolded locally at certain residues. Thedegree of folding is quantified by the fraction ofnative contacts formed, Q, which is used as thereaction coordinate to differentiate betweenesidues between native and 0.8 M NaCl (blue points) andith the most significant shifts in NaCl and Gdn-HCl are
folded, partially folded and unfolded ensembles(see Materials and Methods).39 Here, the nativeensemble is defined as configurations surroundingthe native free energy minima (Qw0.9) between0.6!Q!1.4 To determine whether a simple Ca-onlymodel of the IL-1b protein can capture changes in
Native Energy Landscape for IL-1bthe native IL-1b ensemble at 0.8 M Gdn-HCl,molecular dynamics (MD) simulations were run.In order to approximate the destabilizing propertiesof Gdn-HCl, simulations were run at the foldingtemperature TfZ550 K. This folding temperature isobtained using the minimalist parameters shown inMaterials and Methods, which, if scaled appro-priately, can give the actual experimental foldingtemperature (Tfw335 K).
13 While this simulation Tfmay appear unrealistic, previous studies haveshown that the same str...