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 solv
Trang 1P R I O R I T Y P A P E R
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 (PrPC) [7].On account of this observation, a physico-chemical basis for prion-related molecular diseases is proposed
M A T E R I A L S A N D M E T H O D S
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- and b-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 q¼ q(t) and displayed in Fig.1A for the Duan–Kollman trajectory [6], while its dispersion across all HBs formed at each time,
r¼ 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) FEBS 2002 doi:10.1046/j.1432-1033.2002.03116.x
Trang 2of 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
R E S U L T S
Extent of HB desolvation in folding proteins
Taken together, Fig.1A–C reveal that the extent of HB
desolvation q¼ 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 (CHi, 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 q¼ 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 q¼ 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 q¼ 3.7 and the average dispersion is r¼ 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) in extent of HB-desolvation for a 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 (%)
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].
Trang 3presence 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 q¼ 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 PrPCfiPrPSc
transition
Consistent with results 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 PrPCs 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 Thr216-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 (PrPSc)
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.
Trang 4On 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
R E F E R E N C E S
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 , von Schroetter, 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.