Computational studies of the structure, dynamics and native content of amyloid-like fibrils of...

proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

Computational studies of the structure,dynamics and native content of amyloid-likefibrils of ribonuclease AGiorgio Colombo,1* Massimiliano Meli,1 and Alfonso De Simone2

1 Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco, 9, 20131 Milano, Italy

2Dipartimento delle Scienze Biologiche, Sezione Biostrutture, Universit�a di Napoli Federico II, Via Mezzocannone 16,

I-80134 Naples, Italy

INTRODUCTION

The self-assembly of proteins and peptides into ordered amyloid

fibrils is associated with the evolution of more than 20 syndromes

in humans and animals.1–6 The conversion to amyloid aggregates

is however not restricted to disease-related sequences but appears

to be a general property of polypeptides under appropriate condi-

tions.7–11 Amyloid fibrils share a number of similarities that

include a common cross-b diffraction pattern typical of an elon-

gated stack of b-strands perpendicular to the fibril axis and b-sheets parallel to the axis.

Despite important progress,12–17 the fibrous nature of the

aggregates has hampered the experimental characterization of

many fundamental biophysical properties of the amyloid

state,17,18 and important issues regarding the structural and dy-

namical properties of polypeptides in fibrils are still open. More-

over, there is little consensus on whether native-like domains are

conserved or refolded in the final amyloid state and whether these

domains may retain their catalytic activity.

In this context, Sambashivan et al.19 showed that a designed

amyloid-like fibril of ribonuclease A (RNase A) could contain

native-like molecules capable of enzymatic activity. The amyloid

fibril was engineered starting from the structure of the C-terminal

domain swapped RNase A dimer, expanding with an amyloido-

genic segment (GQ10G) the hinge loop (residues 112–115) con-

necting the core domain (residues 1–111) with the swapped do-

main (residues 116–124). In this model, the C-terminal strand of

each RNase A monomer breaks its noncovalent connections to the

core domain, exposing the Q10 segment, and the C-terminal

strand of another monomer swaps in to take its place. The Q10

stretches from two domain-swapped monomers form an antiparal-

lel b-sheet, which is then complemented by an analogous antipar-

allel b-sheet formed by two more domain-swapped monomers.19

Self-recognition of proteins is completed by precise complementation

The Supplementary Material referred to in this article can be found online at http://www.

interscience.wiley.com/jpages/0887-3585/suppmat/

*Correspondence to: Giorgio Colombo, Istituto di Chimica del Riconoscimento Molecolare,

CNR, Via Mario Bianco, 9, 20131 Milano, Italy. E-mail: [email protected]

Received 20 March 2007; Revised 11 May 2007; Accepted 23 May 2007

Published online 5 September 2007 in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/prot.21648

ABSTRACT

The characterization at atomic resolution of amyloid-

like protein aggregates is one of the fundamental

problems of modern biology. In particular, the ques-

tion whether native-like domains are retained or com-

pletely refolded in the amyloid state and the identifi-

cation of possible mechanisms for macromolecular or-

dered aggregation represent major unresolved puzzles.

To address these issues, in this article we examine the

stability, dynamics, and conservation of native-like

properties of several models of a previously designed

amyloid-like fibril of RNase A (Sambashivan et al.,

Nature 2005; 437:266–269). Through the use of molec-

ular dynamics (MD) simulations, we have provided

molecular-level insights into the role of different parts

of the sequence on the stability of fibrils, the collective

properties of supramolecular complexes, and the pres-

ence of native-like conformations and dynamics in

supramolecular aggregates. We have been able to

show that within the fibrils the three-dimensional

globular domain-swapped units preserve the confor-

mational, dynamical, and hydration properties typical

of the monomeric state, providing a rationalization

for the experimentally observed catalytic activity of

fibrils. The nativeness of the globular domains is not

affected by the amyloidogenic stretches, which deter-

mine the molecular recognition process underlying

aggregation through the formation of a stable steric

zipper motif. Moreover, through the study of the

hydration features of a single sheet model, we have

been able to show that polyglutamine stretches of the

domain-swapped ribonuclease tend to minimize the

interaction with water in favor of sidechain–sidechain

interactions, shedding light on the factors leading to

the supramolecular assembly of b-sheet layers into dry

steric zippers.

Proteins 2008; 70:863–872.VVC 2007 Wiley-Liss, Inc.

Key words: amyloid formation; fibrils; folding mis-

folding; molecular recognition; molecular dynamics;

aggregation.

VVC 2007 WILEY-LISS, INC. PROTEINS 863

of the Q10 side chains stacking in the so-called zipper

spine motif.17

This model was used to explain the conservation of

RNase A enzymatic activity in the fibrillar state through

the formation of complemented active sites via domain-

swapping, implying in turn that fibrils can contain

native-like functional units. However, an experimental

atomic resolution structure is not yet available so that

one has to turn to computational-theoretical methods to

investigate the stability, dynamics, and conservation of

native-like properties of the globular domains in the

fibril model.

To obtain atomic-level resolution insights into these

factors, we have used atomistic molecular dynamics

(MD) simulations in explicit water of several models of

different sizes of the designed RNase A fibrils. We focus

on the role of different parts of the sequence on the sta-

bility of aggregates, the collective properties of supramo-

lecular complexes, and the presence of catalytically active

conformations and motions. Moreover, we analyze the

(de)hydration properties of the cross-b-spine motif and

of the globular domains to gain information on the fac-

tors leading to the supramolecular assembly of b-sheetlayers into dry steric zippers, observed in X-ray studies of

related amyloidogenic sequences.

MATERIALS AND METHODS

The starting structures for the all-atom MD simula-

tions of Q10 RNase A were taken from the Protein Data

Bank with accession numbers 2APQ and 2APU.19 The

former, consisting of a domain-swapped dimer was used

as a starting structure for simulation f2, the latter, con-

taining a model of a whole fibril, was used to construct

the starting structures for simulations f4, f8, and f16.

In all simulations, to mimic proper solution condi-

tions, Lysine amino groups were considered protonated,

while the carboxyl groups were considered to bear a neg-

ative charge. The systems were solvated in a parallel-

piped-shaped box large enough to contain 0.6 nm of sol-

vent around each aggregate. For the single sheet octamer

simulation, the distance between the protein and the box

edge was extended to 1.2 nm. The simple point charge

(SPC) water model was used20 to solvate each protein in

the simulation box. The SPCE flexible water model was

used for the single sheet octamer. Each system was subse-

quently energy minimized with a steepest descent

method for 5000 steps. The calculation of electrostatic

forces utilized the PME implementation of the Ewald

summation method. The LINCS21 algorithm was used to

constrain all bond lengths. For the water molecules, the

SETTLE algorithm22 was used. A dielectric permittivity,

E 5 1, and a time step of 2 fs were used. All atoms were

given an initial velocity obtained from a Maxwellian dis-

tribution at the desired initial temperature of 300 K. The

density of the system was adjusted to perform the first

equilibration runs at NPT (constant Number of Particles,

Pressure, Temperature) condition by weak coupling to a

bath of constant pressure (P0 5 1 bar, coupling time sP5 0.5 ps).23 In all simulations, the temperature was

maintained close to the intended values by weak coupling

to an external temperature bath23 with a coupling con-

stant of 0.1 ps. The proteins and the rest of the system

were coupled separately to the temperature bath. Table I

summarizes the simulation conditions and the number of

atoms for each simulation.

In all cases, the proteins were simulated at 300 K for

50 ns, and all simulations were run at NPT conditions.

All simulations and analysis were carried out using the

GROMACS package (version 3.3),24,25 using the GRO-

MOS96 43A1 force field.26,27 All calculations were per-

formed on clusters of PCs, with Linux operating system.

Graphical display of structures was done using the

PyMOL software.

Water density function calculation

Our hydration analysis is largely based on the solvent

density map whose maxima are assumed to be the mo-

lecular dynamics hydration sites (MDHS).28 The space

surrounding the protein is divided in two shells: the first

shell describes the water within a distance of 0.6 nm

from the protein surface. The second shell extends from

0.6 to 0.8 nm from the protein surface and represents the

bulk solvent shell. The solvent density calculation is grid

based (step-size 0.05 nm). To eliminate protein transla-

tion and rotation, the coordinates of each frame are

transformed by superimposing the current model onto a

reference one. To prevent sweeping effects due to back-

bone flexibility, a selection of frames based on Ca-RMSD

is adopted; therefore, only structures with a Ca-RMSD,

from the reference set, lower than the cutoff of 0.09 nm

are considered.

Time autocorrelation functionand residence time

The time autocorrelation function P(s) is adopted to

provide the residence time of a given interaction. The

function is calculated for each residue to evaluate interac-

Table ISummary of Simulation Conditions and Number of Atoms

SimulationNumberof chains

Numberof atoms

Solventmolecules

Length(ns)

f2 2 36,590 1280 50f4 4 61,885 8795 50f8 8 97,991 28,997 50f16 16 151,572 42,525 50

G. Colombo et al.

864 PROTEINS DOI 10.1002/prot

tions with the remaining residues or with water. The for-

mula is

PðsÞ ¼X

t

d W ðtÞ;W ðt þ sÞð Þ

where the delta function d(W(t), W(t 1 s)) assigns 0 or

1, respectively, whether the indexes of the partners at

times t and t 1 s differ or not. The resulting time auto-

correlation function is then fitted by an exponential

model.

RESULTS

The different fibril models analyzed herein consist of

2, 4, 8, and 16 chains. Studying constructs of increasing

dimensions may yield information on the properties of

growing aggregates. In the models with 2 and 4 chains, a

single b-sheet is formed by the alignment of Q10 amyloi-

dogenic segments from domain swapped RNase A mole-

cules. In the models with 8 and 16 chains, a full cross-b-spine formed by the juxtaposition of two b-sheets is con-sidered (Figure 1, Table I). All the MD runs used for

analysis are 50 ns long and are labeled according to the

number of protein chains in the fibril model. Simulations

f2, f4, f8, and f16 are thus run on models consisting of

two, four, eight, and sixteen chains, respectively. One

more simulation of a single sheet of eight monomers was

run to analyze the possible role of hydration in the

aggregation mechanism.

Structural and dynamical properties

Figure 2(a) shows the time evolution of the Ca atom

root mean square deviation (RMSD) of the simulated

structures with respect to the initial X-ray based model.

The RMSD calculated over all the Ca’s of the proteins in

each construct increases in time with an inverse propor-

tionality to the number of chains in the system: simula-

tion f2 reaches the highest value, followed by f4, f8, and

f16.

Restricting the RMSD calculation only to the Q10 seg-

ments of the spine [Fig. 2(b)] shows that these regions

remain close to the starting geometry for simulations f4

to f16. In f4, the RMSD initially increases to around 0.3

nm followed by a decrease to 0.17 nm. In the cases of f8

and f16, the steric zipper RMSD value is consistently sta-

ble around 0.17 and 0.15 nm for the whole simulation

time. Only in the case of f2 does the RMSD of Q10

diverge to values as high as 0.5 nm due to the high flexi-

bility characterizing the dimer. Large quaternary structure

fluctuations were already noted in the case of the original

Figure 1Structures of the protofibril simulated models. f2 and f4 are a single b-sheet formed by the alignment of Q10 amyloidogenic segments from domain swapped RNase A

molecules. In f4 the Q10 segment is displayed using the representation of van der Waals radii of the atoms. In the models with 8 and 16 chains, f8 and f16, a full cross-b-spine is considered. In f16 the Q10 segment is displayed using the representation of van der Waals radii of the atoms. [Color figure can be viewed in the online issue,

which is available at www.interscience.wiley.com.]

MD Simulations of RNase A Fibrils

DOI 10.1002/prot PROTEINS 865

C-terminal domain-swapped dimer of RNase A,29 and

they are mostly due to the lack of structural constraints

imposed by the presence of other monomers in the pro-

tofibril. The average internal RMSD for each globular do-

main amounts to 0.15 nm in all simulations. These data

suggest that the 3D native structures of globular domains

are preserved, while their orientations at the end of the

simulations are different from the initial positions.

Analysis of the flexibility in terms of residue-based

root mean square fluctuations (RMSF) shows minimal

values for the Q10 stretches compared with the fluctua-

tions of the other regions of the proteins making up the

fibrils in simulations f4, f8, and f16 (Fig. 3). As far as the

globular domains are concerned, the residue-based RMSF

values for each subunit are comparable to those already

observed for simple domain-swapped dimers lacking the

Q10 expansion, suggesting that the fluctuations and

breathing motions of the native-like part of the protofi-

bril linked to reactivity are conserved in bigger aggre-

gates. In the case of f2, the steric zipper region displays

relatively higher values. The flexibility of the steric zipper

region appears to decrease as the fibril’s dimensions

increase. The stacking of multiple amyloid-forming seg-

ments from different identical RNAse A molecules stabil-

izes and rigidifies the extended antiparallel b-sheet by

locking the sterically complementary alkyl sidechains of

the interface glutamines via van der Waals and hydrogen

bonding interactions17 (amide stacks). The number of

hydrogen bonding interactions due to amide stacks

within one b-sheet (all models from f2 to f16) remains

constant during the simulation time. The same holds for

the calculation of the number of contacts between two

juxtaposed sheets in the cross-b-spine (f8 and f16) (see

supplementary material, Figs. S1–S4). The presence of

these intersheet interactions determines further stabiliza-

tion and a consequent rigidification of the final structure

of the spine of the fibril. These observations are consist-

ent with the results obtained in the analysis of cross-b-spine stabilization in fibrils of GNNQQNY peptides.17,30

Interestingly, these interactions do not perturb the

folding of the remaining globular domains. The analysis

of structural parameters such as the time evolution of

secondary structure and the time evolution of contact

maps (see supplementary material, Figs. S1–S4) demon-

strates the absence of major conformational changes or

unfolding events within each single native-like subunit.

This set of data shows that while the zipper spine

region is highly ordered, stable, and relatively rigid, the

native-like swapped domains dangle in solution, protrud-

ing from the fibril spine. The orientations of the globular

domains during the MD simulations are thus variable

and different from the starting structures. These rigid

body motions would prevent the observation of coherent

X-ray diffractions for the globular domains. The only

part of the aggregate capable of generating reflections

would thus be the spine, leading to the observed X-ray

diffraction pattern typical of fibrils.

Collective properties of the fibril

The differential dynamics of the globular domains and

of the cross-b-spine motif in the supramolecular assem-

blies raises the question whether it is possible to identify

collective, ordered motions as in normally folded pro-

teins, or whether native-like parts fluctuate randomly in

solution. To address this question, the covariance matri-

ces of displacements of pairs of Ca’s were calculated from

each 50 ns trajectory from simulations f4 to f16 and ana-

lyzed with the essential dynamics (ED) method.31 ED

Figure 2Time evolution of the root mean square deviation from the initial structure of

all the atoms in the fibrils (a) and of the Q10 segment only (b). The black line

indicates f16, red f8, green f4, and blue f2.

G. Colombo et al.


identifies the physically relevant (essential) protein

motions represented by the eigenvectors of the covariance

matrix associated to the largest eigenvalues. These eigen-

vectors describe the most significant modes of conforma-

tional motion of the molecules in the 3D space. The first

three eigenvectors correspond to 75% of the overall eigen-

values for f4 and f8 and to 60% for f16. In all cases, the

main motion involves the globular domains, with very lit-

tle or no contribution from the cross-b-spine residues.

This mode represents a concerted clockwise twisting of

two globular domains involved in domain swapping with

each other [Fig. 4(a,b)]. The residues participating mostly

to this motion (most mobile) are shown in the figure with

blue arrows representing the magnitude of the motion.

The highest displacements involve the globular domains.

Observation along the cross-section of the protofibril sug-

gests that this motion will impose a left-handed twisting

on the fibril, consistent with previous observations on the

spontaneous twisting of model protofibrils of smaller pep-

tides.30,32 The second and third modes involve transla-

tions of the globular domains and a slight flattening

motion of the Q stretches of the steric zipper region.

Moving along the axis of the fibril, no correlation of

the motions of the globular domains is observed. The

residue-based fluctuations (RMSF) obtained after projec-

ting the trajectories on the main eigenvectors are consist-

ent with these observations [Fig. 4(d)]. In the projection

on the first main eigenvector, only residues belonging to

the globular domains experience significant fluctuations,

while in the second and third modes the fluctuations of

the steric zipper residues display higher intensities. The

catalytic histidines (H) undergo minor fluctuations along

the three main eigenvectors. These residues belong to

different complementary molecules and are coupled

through the domain swapping process, which eventually

leads to the formation of a catalytically active complex.

The distances between histidines of the complemented

active sites present in the X-ray model are in general

conserved during the simulations. In simulation f4, the

active site formed by chains 3 and 4 is initially disrupted

but is reformed at the end of the simulation. The same

happens for chains 3–4 and 7–8 in simulation f8. In sim-

ulation f16, all the original complemented active site geo-

metries are maintained, except for the case of the active

site formed by the N-terminal part of chain 3 and the C-

terminal part of chain 4. The histidine belonging to the

latter chain is however replaced by the C-terminal histi-

dine of chain 10. The growth of the fibril does not

Figure 3Residue based RMSF calculated on the whole 50 ns of the trajectory. The circles identify the regions occupied by Q10 stretches. In f4, f8, and f16, the fluctuations of the

Gln amyloidogenic insertion assumes a minimum value.



appear to negatively influence the determinants of enzy-

matic activity.

Interestingly, the principal component for the single

globular units [Fig. 4(c)] show the breathing motions of

the b-sheet regions, which have been identified as funda-

mental for the catalytic activity of the protein in mono-

meric RNase A, C-terminal, and N-terminal domain-

swapped dimers.29,33

Fibril hydration

The analysis of the hydration properties of a single

sheet octamer (representing half of the protofibrillar

unit) was used to investigate the role of water in the for-

mation of a full protofibril. The water density map was

calculated from the atomic coordinates of the protein

and solvent (see methods) collected during a 10-ns simu-

lation; peaks of the density function represent MDHS.28

Remarkable features immediately appear. A uniform dis-

tribution of MDHS is detected on the RNase A globular

domains, whereas the cross-b spine forming region

appears asymmetrically solvated. It has to be noted that

the single sheet presents a uniform plate of Gln side-

chains (polyQ plate) [Fig. 5(a–c)]. In particular, the Q10

surface not involved in the formation of the fibril dis-

plays stable MDHS and appears as a normally hydrated

hydrophilic surface [Fig. 5(a)]. We will refer to this sur-

face as ‘‘internal’’ since it faces the region where the glob-

ular moieties are placed. In contrast, the opposite surface

(‘‘external surface’’), which is supposed to engage the dry

interface in the double layer formation, does not display

any stable MDHS [Fig. 5(b,c)]. Here, in spite of the

hydrogen bonding properties of glutamines, water is

unable to find fixed and stable anchoring points and

undergoes very fast exchange with the bulk phase (see

residence time Table II); as a result this surface appears

on the average to be ‘‘dewetted.’’

Such peculiar behavior is due to sequence and struc-

tural aspects. On average, each Gln of the plate is sur-

rounded, in a 2D network, by four very accessible Gln

neighbors: two stacking on the flanking strands and the

two contiguous Gln on the same strand. This structural

arrangement determines a high crowding of Gln side-

chains, which are favored to interact with one another

through van der Waals packing of the alkyl part of the

side chain and through a complex dynamic network of

Figure 4The directions of the main displacements along the first eigenvector of the covariance matrix are shown as arrows of equal length. For the sake of visualization only a slice

of the whole fibril is represented in two different orientations (a,b). The Q10 appears not to participate in this motion, while the globular parts translate and rotate as

rigid bodies. Subpanel (c) reports the essential breathing motions of the globular part of the structure. These motions are very similar to what was previously observed for

simple monomers or dimers of RNase A. Subpanel (d) shows the residue based RMSF after projecting the trajectories along the first main eigenvector of the covariance

matrix. Small triangles indicate catalytic histidines. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

G. Colombo et al.


sidechain–sidechain H-bonds (amide stacks). This has

the effect of subtracting H-bonding donors and acceptors

for protein–water interaction. This crowding and the

consequent intramolecular H-bonding of Gln residues

prevent water molecules to stably localize on the polyQ

surface. Overall the sidechain–sidechain H-bonds have

the effect of disrupting sidechain–water H-bonds and

marginally subtracting H-bonding donors and acceptors

for protein–water interaction (see Table II).

In contrast, the opposite face (internal face) of the

polyQ sheet, although presenting the same 2D network

of Gln, is regularly hydrated because of the surrounding

globular domains. These domains slow down the

exchange between bulk solvent and protein hydration

layer (see residence time Table II). In addition, exposed

residues from the globular domains provide anchoring

points to water molecules, creating an H-bonding net-

work that propagates within the region enclosed by the

polyQ sheet and the globular domains.

Active site hydration

The analysis was extended to the hydration properties

of the globular ribonuclease domains in the fibril. Several

studies have shown that water molecules are important

to preserve the right active site conformation in the

ligand-free form. Brunger et al.34 showed the impor-

tance of Lys7-Lys41 interaction, which is mediated by a

water cluster. Moreover, a comparison between CMP-com-

plexed and ligand-free forms showed that water replaces

the substrate by keeping the active site in the active con-

formation. Our hydration analysis showed that in the

protofibrillar structures analyzed in this work, both the

water cluster that mediates Lys7–Lys41 interaction [Fig.

6(a)] and the pattern of hydration sites that preserves theFigure 5(a) RNase A single sheet octamer solvation map. MD water high-density sites

contoured at 2.5 times the bulk solvent density. The globular domains and the

internal surface appear regularly hydrated, while the external polyQ surface

appears to be dewetted. (b,c) Residence times of sidechain–water Hbonds. The

residence time is evaluated from the time autocorrelation function. The residence

times of Gln’s of the fourth and fifth strand (from the 8-mer single layer) are

reported in panels (b) and (c) respectively. Waters in contact with the external

face are very mobile and exchange rapidly with the bulk solution, whereas

waters in contact with the internal face are slowed down from the globular

moieties. As a result, the residence times present alternate profiles. Even numbers

indicate Gln residues on the external surface, whereas the odd numbers indicate

those on the internal surface. [Color figure can be viewed in the online issue,


Table IIResidence Times and Average Occurrences of Gln-Water and

Gln-Gln Hydrogen Bonds

Internal External

Residence time(ps) H-bonds Gln-H2O 286.04 49.47Average occurrence H-bond Gln-H2O 0.66 1.22

Residence time(ps) H-bonds Gln-Gln 323.73 279.32Average occurrence H-bond Gln-Gln 0.36 0.37

Figure 6Stable hydration sites in the complemented active site of one of the globular

domains of the whole fibril. Hydration is consistent with previous analogous

observations on the monomer.34 [Color figure can be viewed in the online issue,




right active site conformation of His12 and His119 in the

domain-swapped arrangement are conserved [Fig. 6(b)].

This observation helps rationalize the conservation of en-

zymatic activity in full fibrils from RNase A.

DISCUSSION

The variety of dynamic, conformational, and hydration

features observed in our simulations of RNase A rever-

berates the complexity of the free energy surface that

may characterize the aggregated state of fibril forming

proteins.

The residues of the b-sheet region, formed by the Q10

amyloidogenic loop expansion, display low fluctuations

and high conformational rigidity. Highly dense packing

of Gln sidechains, through intrasheet hydrogen bonding

interactions of the amide stacks and intersheet van der

Waals interactions of their alkylic parts in the steric zip-

per, is the main determinant of the stabilization of the

cross-b-spine motif and of the fibrillar structure. These

conformational properties and the presence of strong

hydrophobic interactions in b-strands cores of amyloids

have already been largely recognized as important factors

for the stability of b-amyloid fibrils.35 Interestingly, the

high number of attractive interactions in the limited

space of the steric zipper is also useful to explain the

high resistance of fibrils to mechanical fracture, as

recently observed with force spectroscopy studies of insu-

lin fibrils.18

In contrast, the analysis of the domain-swapped globu-

lar regions reveals that they preserve all the properties

defining a ‘‘functional unit’’ even in the protofibrillar

models. In particular, the three-dimensional fold, the

breathing functional motions necessary for catalytic activ-

ity, the reactive geometry of catalytic sites and the hydra-

tion of critical residues appear to be essentially the same

as those observed for both the monomeric form and the

nonamyloidogenic C-terminal and N-terminal domain-

swapped dimeric form of RNase A.29,33 In terms of col-

lective properties, the globular domains move as rigid

bodies dangling from the central b-sheet, and the

motions of whole supramolecular aggregates appear to

impose a left-handed twisting on the fibril, a phenom-

enon observed previously on model small peptide proto-

fibrils30,32 and in several experimental low-resolution

structures. The rigid-body dangling motions of the glob-

ular domains around the central rigid spine help explain

the difficulties in obtaining coherent diffractions for these

regions of the aggregates. On the other hand, the rigidity

and order in the cross-b-spine motif explain the obser-

vation of typical fibril-like reflections only at 0.48 and

1 nm.

Overall our observations based on long timescale,

explicit solvent MD simulations validate the suggestion

by Eisenberg and coworkers that functional, globular

units can be considered ‘‘native-like’’ in aggregated fibril-

lar states and support the idea that domain swapping

may be considered a mechanism for amyloid formation.

In this context, Liu et al.36 already pointed out a high

degree of parallelism between 3D domain swapping and

amyloid fibril formation. The two phenomena display

the same type of specificity (being formed by only a sin-

gle type of protein), the presence of two (or more) stable

conformational states separated by high-energy barriers

and the possibility to form linear complexes. The pres-

ence of native-like folded states in higher order aggre-

gates was also confirmed recently by Gotte and co-

workers, who identified and partly characterized larger

domain-swapped oligomers of RNase A.37 The nativeness

of the globular domains and of their conformational dy-

namics is dictated uniquely by their sequence, which is

not changed in the aggregate. The aggregation process

and the stabilization of the fibril are driven principally by

the interactions among the Q10 expansions with no per-

turbation of the globular units. In this picture, there is

no need to invoke a complete unfolding and refolding to

a different structure of the whole sequence.

Our simulations and hydration analysis provide molec-

ular level insights into the role of water in fibril forma-

tion and suggest a possible picture of fibrillogenesis

mechanisms. The single layer polyQ sheet appears to be

dually solvated with both a hydrated and a dry surface.

The regularly hydrated face is on the site where globular

ribonucleases moieties are located (internal surface),

whereas the dewetted surface is fully exposed to the sol-

vent (external surface) and is on the side of the double

layer assembly. The same interactions among Gln side-

chains that are responsible for the stabilization of the

fibril are also responsible for favoring intramolecular

Gln–Gln H-bonding interactions, over intermolecular

Gln–water interactions. The net result of our hydration

analysis is that the face of the polyQ single layer that is

eventually engaged in the formation of the steric zipper

is intrinsically dewetted.

This finding is significant in terms of solution proper-

ties of the system. Indeed, tightly bound waters on the

protein surface are connected to high desolvation energy.

Accordingly, regions poorly surrounded by MDHS are

preferentially selected in processes requiring large desol-

vation energies. As a result, dewetted surfaces might

promote hydrophobic collapses38,39 and extended protein–

protein interactions. Finding a dewetted surface, com-

posed of partly polar sidechains, may be considered un-

usual. Noteworthy, this result is in large agreement with

the recent finding that monomeric polyQ forms collapsed

structures.40 Overall these data claim that polyQs have a

hydrophobic behavior, a singular property for a polymer

of an aminoacid considered hydrophilic in the hydropho-

bicity scales41–43; indeed polyQ stretches acquire a pecu-

liar hydrophobilicy that appears to be due to the intra-

molecular crowding of sidechain H-bonds donors and

G. Colombo et al.


acceptors. It is worth noting that we do find a local pro-

tein–water interaction (see Table II); however, the above-

described factors prevent the formation of stable and

long-lived sidechain–water H-bonds. This constitutes the

first rationalization of how the peculiar hydration of

polyQ surfaces makes them good candidates for the dry

steric zipper association.

The analysis of the properties of polyQ stretches may

contribute to the understanding of the role of hydrogen

bonding versus hydrophobic effects in the stabilization of

proteins and protein complexes. Gln residues (as well as

Asn residues) can form triple hydrogen bonds (two

involving main chain groups, one involving the amidic

sidechain moiety), which fit optimally into the antiparal-

lel b-sheet geometry of the polar zipper. In conditions of

thermodynamic control, this would lead to the net stabi-

lization of the state displaying the highest possible num-

ber of these interactions (the aggregate in this case).

Hydrophobic groups (such as the alkyl moieties of Gln,

Asn, or other aliphatic–aromatic side chains), in contrast,

may play a relevant role under kinetic control, imposing

the stereochemical constraints necessary for molecular

recognition in the formation of ordered protein struc-

tures and complexes. In this context, the height of the

barrier for complex formation is determined by the type

of residues present in the sequences and their sequence

and spatial relationships, while the final stability of the

complexes is determined by the net stabilization provided

by protein–protein hydrogen bonds.

To our knowledge, our study represents the first case

in which the properties of a complex, supramolecular ag-

gregate from a whole protein are analyzed with atomistic

methods. We could obtain a first rationalization of the

dynamic, flexibility and hydration properties of the fibril,

showing that the presence of the fibrillogenic region does

not affect the properties of the globular regions, which

preserve their fold and functional motions behaving as

native-like functional units. The extension of this

approach to other constructs of different proteins may

provide important and general information on the deter-

minants of self-organization of biological systems and

suggest ideas for their modification and use in biotech-

nological and medical applications.

ACKNOWLEDGEMENTS

This work was supported by funding under the Sixth

Research Framework Programme of the European

Union (ref. LSHB-CT-2006-037325) under the FIRB

program Folding e aggregazione di proteine: metalli e

biomolecole nelle malattie conformazionali, and under

support of the Ministero degli Esteri exchange program

Understanding the Molecular Determinants of Amyloid

Fibril Formation in Human Neurodegenerative Diseases.

The authors gratefully acknowledge Prof. David Eisen-

berg and his coworkers for helpful discussion and com-

ments. The authors also acknowledge Dr. Giacomo Car-

rea for support.

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