Báo cáo khoa học: Insufficient hydrogen-bond desolvation and prion-related disease ppt

PRIORITY PAPER Insufficient hydrogen-bond desolvation and prion-related disease Ariel Ferna ´ ndez* Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA A structuring and eventual exclusion of water surrounding backbone hydrogen bonds takes place during protein fold- ing as hydrophobic residues cluster around such bonds. Taken as an average over all hydrogen bonds, the extent of desolvation is nearly a constant of motion, as revealed by re-examination of the longest all-atom trajectory with explicit solvent [Y. Duan & P. A. Kollman (1998) Science 282, 740]. Furthermore, this extent of desolvation is pre- served across native soluble proteins, except for cellular prion proteins. Thus, a physico-chemical picture of prion- related disease emerges. The epitope for protein-X binding, the region undergoing vast conformational change and the trigger and locker for this change are inferred from the location of under-desolvated hydrogen bonds in the cellular prion protein. Keywords: protein folding; hydrogen bond; backbone desolvation; all-atom trajectory; prions. The progressive structuring, immobilization and ultimate removal of water surrounding the backbone hydrogen bonds (HBs) of a protein turn the latter into major determinants of protein folding and structure [1–5]. However, to the best of my knowledge, a systematic examination of evolving environments surrounding back- bone HBs has been lacking. The inherent stability of such bonds is essentially defined by the solvation free energy of the unbound reference state with its exposed backbone polar groups, the amides and carbonyls [1–3]. Thus, most of HB stability is brought about by the destabilization of the unbound state due to progressive removal of surrounding water from the backbone polar moieties. The inaccessibility of HBs to solvent takes place as the protein places hydrophobes around its backbone polar groups [1] during the folding process. This backbone burial induces HB formation as a means to compensate for the unfavorable desolvation of the backbone polar moieties [3]. In this regard, a question arises and is addressed herein: what are the most effective ways for a protein to cluster hydrophobes in order to protect its backbone HBs? Here I approach this question by investigating the packing of HBs in the longest available all-atom molecular dynamics (MD) trajectory, the 1 ls run for the autonomous folder villin headpiece [6], systematically keeping track of the environments surrounding each HB. The work reported seeks to establish a pervasive desolvation motif in the folding process. My re-examination of the Duan–Kollman trajectory [6] reveals a nearly constant average extent of HB desolvation or average number of surrounding hydro- phobes with relatively small dispersion across all HBs in the chain, supporting the existence of a constant of motion. Furthermore, this average extent of HB desolvation appears to be a constant across native protein structures, as a direct examination of a large sample of the PDB reveals. The second part of this paper is devoted to a comple- mentary question: what is the structural significance of under-desolvated hydrogen bonds (UDHBs) and which proteins are definite outliers within the distribution of average extents of HB desolvation? An analysis of the PDB reveals that the only soluble proteins with an inordinately large number of UDHBs are the cellular prion proteins (PrP C ) [7]. On account of this observation, a physico- chemical basis for prion-related molecular diseases is proposed. MATERIALS AND METHODS First, I systematically inspected the desolvation patterns of backbone HBs along a folding trajectory. To do this, I define two ellipsoids for a backbone HB with major radius R fixed at 7 A ˚ and foci at the a-andb-carbons of the residues paired by the HB. The inferences made in this section are robust to moderate changes in the desolvation radius, holding within the range R ¼ 7.0 ± 0.3 A ˚ . An amide-carbonyl HB is determined by an N-O distance within the range 2.6–3.4 A ˚ (lower and upper bounds of typical bond lengths) and a latitude of 45 degrees in the N-H-O angle. As a next step, I counted the number of hydrophobic (third-body) residues whose a-carbon is contained within the HB desolvation ellipsoids. The counting includes the residues paired by the HBs themselves if they happen to be hydrophobic. Thus, the average number of third-body hydrophobes per HB constitutes a measure of the extent of HB desolvation. This quantity is denoted by q ¼ q(t) and displayed in Fig. 1A for the Duan–Kollman trajectory [6], while its dispersion across all HBs formed at each time, r ¼ r(t), is displayed in Fig. 1B: the latter is never larger than 30% of the mean value. Figure 1C shows the number Correspondence to A. Ferna ´ ndez, Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, USA. Fax: + 1 773 7020439, E-mail: ariel@uchicago.edu Abbreviations: HB, hydrogen bond; UDHB, under-desolvated hydrogen bond. *On leave from Instituto de Matematica, Universidad Nacional del Sur, CONICET, Bahia Blanca 8000, Argentina. (Received 27 May 2002, accepted 8 July 2002) Eur. J. Biochem. 269, 4165–4168 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03116.x of backbone HBs formed plotted against the number of three-body correlations, i.e. number of third-bodies desolvating hydrogen-bonded pairs of residues. Note that a single hydrophobe might be engaged in more than one three-body correlation. RESULTS Extent of HB desolvation in folding proteins Taken together, Fig. 1A–C reveal that the extent of HB desolvation q ¼ 5 is very nearly a constant of motion for the folding trajectory. With the caveat that there are more accurate ways to define the extent of HB protection, i.e. carbonaceous (CH i , i ¼ 1,2,3) groups contained in the desolvation ellipsoids, whose average number remains in the range 15.0 ± 1.8, the results presented here imply the existence of an elementary motif preserved throughout the folding process. Not only is q ¼ 5 nearly a constant of motion in protein folding: a direct inspection of 355 native folds from the protein data bank revealed that 97% of the autonomously folded and soluble proteins of different sizes examined (33 < N < 401) have q in the range 5.00 ± 0.23, with a dispersion r invariably lower than 18%. These statistics are illustrated by a description of 20 proteins the upper part of Table 1. This observation suggests that the same building constraints present in the native folds govern the entire folding process. Proposed physico-chemical basis of prion desease The soluble cellular prion proteins [7,8], 10 of which are quoted in the lower part of Table 1, are definite outliers to the q ¼ 5 constraint, and in fact they are the only outliers among soluble proteins in the entire sample of the PDB. Their average extent of HB desolvation is q ¼ 3.7 and the average dispersion is r ¼ 22%. These statistics signal the Table 1. Number of hydrophobe-HB 3-body correlations (C 3 ), number of backbone hydrogen bonds (Q), average extent of HB-desolvation q ¼ C 3 /Q and Gaussian dispersion (r)inextentofHB-desolvationfora sample of soluble proteins from the PDB (upper part) and for definite outliers to the q ¼ 5 building constraint (lower part). PDB accession no. C 3 Q q r (%) 1aa2 257 52 5.04 10.18 1lou 242 47 5.15 13.05 1ris 230 45 5.11 12.87 1aue 250 49 5.10 11.80 256b 394 75 5.25 16.05 1ubi 155 31 5.00 10.06 1gb4 80 16 5.00 10.14 1srl 40 8 5.00 12.83 2ptl 74 16 4.62 16.33 1crc 136 28 4.85 9.60 1vii 30 6 5.00 12.55 1hhh 446 86 5.18 12.68 1mim 318 64 4.96 17.62 1ifb 215 43 5.00 8.83 1hhg 468 95 4.92 11.09 1e4j 225 45 5.00 12.11 1e4k 233 46 5.07 11.15 1gff-1 612 124 4.93 11.58 1csk-A 111 22 5.04 12.01 1c3t 105 21 5.00 10.78 1dxo 215 59 3.64 21.8 1dwy 219 59 3.71 22.1 1dwz 216 60 3.60 24.2 1b10 233 58 4.01 21.3 1qlx 228 58 3.93 19.6 1qlz 210 53 3.96 20.6 1qmo 216 57 3.79 20.2 1qm1 210 56 3.75 21.4 1qm2 199 57 3.49 24.1 1qm3 196 53 3.70 22.7 Fig. 1. (A) Time-dependent average extent of protection of the backbone HBs, q ¼ q(t); (B) dispersion, r(t), of hydrophobic cluster sizes; and (C) number of backbone HBs plotted against number of 3-body correlations. All plots resulted from a re-examination of the Duan-Kollman MD trajectory [6]. 4166 A. Ferna ´ ndez (Eur. J. Biochem. 269) Ó FEBS 2002 presence of an inordinately large number of UDHBs. An UDHB is here operationally defined as one surrounded by at most two hydrophobes. These UDHBs, being highly solvent-exposed, are indeed weak bonds. The UDHBs in native folds are displayed in Fig. 2 for a hemoglobin b-subunit (pdb.1bz0, chain B), a representative protein of the common q ¼ 5 building constraint, and for a definite outlier, the human prion protein pdb.1qm1 [8]. The desolvated (q > 2) backbone HBs are represented as gray segments joining the a-carbons of the paired residues, while the green segments represent UDHBs. The a-carbon virtual-bond backbone is displayed as a red line and the yellow and gray spheres indicate a-carbons, respectively, associated with overexposed (under 33% buried) and buried hydrophobic residues. Significantly, the hemoglobin b-sub- unit (Fig. 2A) contains only two UDHBs (Thr4-Lys8 and Pro5-Ser9), strikingly located next to the residue Glu6, whose mutation into Val6 is known to cause sickle cell anemia. In soluble proteins with q close to 5, the number of UDHBs is never larger than 12% of the total number of backbone HBs. In contrast, the number of UDHBs in the cellular prion proteins is approximately 40% of the total number of backbone HBs (24 out of 56 HBs for 1qm1, and 23 out of 57 HBs for 1qm2). Because UDHBs are very vulnerable to water attack, these statistics signal an unstable conformation that must undergo major structural rear- rangement, as is indeed the case [7,8]. Insufficiently desolvated HBs and the PrP C fiPrP Sc transition Consistent withresults reported previously [7,8], the a-helix 1 is the most susceptible to structural change into b-strand and condensation onto the adjacent b-sheet nucleus. This is so as in all examined prion proteins 50% to 80% of the HBs in helix 1 are actually UDHBs (Figs 2B and 3). At this point we may rationalize the transformation from cellular to scrapie-like conformation by analyzing the pattern of UDHBs in the prototypical prion protein from Syrian hamster [7] (pdb.1b10, Fig. 3). The residues known to be involved in prion trasmission Trp145, Arg148 happen to be paired by UDHBs, and thus, their engagement in binding is triggered by the possibility of providing further desolvation to the UDHBs. On the other hand, hydropho- bic residues 138, 139 and 141, if exogenously paired, as they are when engaged in prion transmission, would no longer effectively desolvate the HBs from helix 1. The resulting overwhelmingly large number of UDHBs on helix 1 (now five out of a total of six) would cause it to dismantle, thus inducing the conformational transition. The helix 1 in human PrP C s 1qm1 and 1qm2 is even more susceptible to be dismantled as its ratio of UDHBs to HBs is even higher (cf. Fig. 2B). This conformational change leading to a condensation onto the existing nucleating b-sheet finds a thermodynamic compensation: the stabilization of the UDHBs Thr216- Lys220 and Lys220-Ala224 in helix 3 brought about by the purported proximity of hydrophobic residues 138, 139 and 141 (Fig. 3). Thus, the desolvation of these two UDHBs should be regarded as the ÔlockingÕ mechanism for the purported b-sheet in the scrapie form (PrP Sc ). Fig. 3. HB-pattern for the Syrian hamster prion protein (pdb.1b10). Fig. 2. (A) HB-pattern for the b-subunit of hemoglobin (pdb.1bz0, chain B) and (B) HB-pattern for the human cellular prion potein pdb.1qm1. Ó FEBS 2002 Hydrogen-bond dehydration in prions (Eur. J. Biochem. 269) 4167 On the other hand, a mutation of the helix 1 desolvator Phe141 into a polar residue is predicted to increase substantially the probability of structural transition into the scrapie form, as such a mutation decreases the extent of desolvation of the nearby Glu146-Tyr150, Asp147-Arg151 HBs in helix 1, turning them into UDHBs (in the w.t., Phe141 lies within their desolvation spheres). Thus, as a result of the mutation, helix 1 increases the number of UDHBs from three to five. This transformation would induce water attack on this part of the structure leading to its dismantling. The residues Gln168, Gln172, Thr215 and Gln219 clearly form the binding epitope with protein X, known to be located in helix 3 and the 167–171 looped region [8]. These residues are easily identified as paired by UDHBs (Fig. 3). Thus, there is a considerable thermodynamic benefit (i.e. the added stabilization of the UDHBs) resulting from the exogenous desolvation of the partici- pating UDHBs. This extra desolvation is brought about by association of the cellular prion protein with the purported protein-X partner. The two C-terminal UDHBs are mere artifacts since the C-terminus is highly flexible, and thus do not signal binding sites. This statement is consistent with the C-terminal fraying revealed by the proton exchange protection factors [8]. The remaining UDHBs located at helix-loop junctures are needed to add the flexibility to these regions, as required during the conformational transitions. REFERENCES 1. Ferna ´ ndez, A., (2002) Time-resolved backbone desolvation and mutational hot spots in folding proteins. Proteins 47, 447–457. 2. Vila, J.A., Ripoll, D.R. & Scheraga, H.A. (2000) Physical reasons for the unusual alpha-helical stabilization afforded by charged or neutral polar residues in alanine-rich peptides. Proc. Natl Acad. Sci. USA 97, 13075–13079. 3. Makhatadze, G. & Privalov, P.A. (1995) Energetics of protein structure. Adv. Protein Chem. 47, 307–425. 4. Baldwin, R.L. (2002) Protein folding: Making a network of hydrophobic clusters. Science 295, 1657–1658. 5. Krantz, B.A., Moran, L.B. Kentsis, A. & Sosnick, T.R. (2000) D/H amide kinetic isotopic effects reveal when hydrogen bonds form during protein folding. Nature Struct. Biol. 7, 62–71. 6. Duan, Y. & Kollman, P.A. (1998) Pathways to a protein folding intermediate observed in a 1ls simulation in aqueous solution. Science 282, 740–744. 7. Prusiner, S.B. (1998) Prions. Proc. Natl. Acad. Sci. USA 95, 13363– 13383. 8. Zahn,R.,Liu,A.,Luhrs,T.,Riek,R.,vonSchroetter,C.,Lopez- Garcia, F., Billeter, M., Calzolai, L., Wider, G. & Wuthrich, K. (2000) NMR solution structure of the human prion protein. Proc. Natl. Acad. Sci. USA 97, 145–150. 4168 A. Ferna ´ ndez (Eur. J. Biochem. 269) Ó FEBS 2002 . PRIORITY PAPER Insufficient hydrogen-bond desolvation and prion-related disease Ariel Ferna ´ ndez* Institute for Biophysical. physico- chemical basis for prion-related molecular diseases is proposed. MATERIALS AND METHODS First, I systematically inspected the desolvation patterns of backbone