The Native Energy Landscape for Interleukin-1β. Modulation of the Population Ensemble through Native-state Topology

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  • Native-state Topo

    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: pajennin@ucsd.edu

  • 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.

    Results

    Experimental measurements of the native basin

    Fluorescence spectroscopy

    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 (res