Báo cáo khoa học: Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach ppt

In the majority of the proteins whose molten globule states have been characterized, both by experimental and the-oretical methods, predominantly a-helical secondary structure persists i

retinol-binding protein using a molecular dynamics

simulation approach

Emanuele Paci1, Lesley H Greene2, Rachel M Jones2and Lorna J Smith2

1 Institute of Molecular Biophysics, School of Physics and Astronomy, University of Leeds, UK

2 Department of Chemistry and Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, University of Oxford, UK

The detailed characterization of molten globule states

of proteins continues to be an area of intense research

activity and interest (for reviews see [1,2]) This is in

part because these equilibrium partially folded states

have been found to have many similarities to kinetic

protein folding intermediates As such, the properties

of these states can therefore give important insights

into the determinants of protein structure and folding

[2] Molten globule states of proteins are also

postula-ted to be involved in a range of important

physiologi-cal processes, including the insertion of proteins into

membranes, the release of bound ligands and

aggrega-tion [1] In this latter area, molten globule-like species

are thought in some systems to be precursors of amy-loid fibril formation [3,4]

Molten globule ensembles are characterized by hav-ing a pronounced amount of secondary structure, in a compact state that lacks most of the specific tertiary interactions coming from tightly packed side chain groups [1,2] One of the molten globule states that has been studied in the most depth is that of a-lactalbumin [5] In this case, it has been possible using nuclear magnetic resonance (NMR) methods to gain a residue specific picture of the noncooperative unfolding of the molten globule during denaturation with urea [6–8]; data from these experiments have also been used as

Keywords

lipocalin; molecular dynamics; molten

globule; protein folding; retinol-binding

protein

Correspondence

L J Smith, Department of Chemistry,

University of Oxford, Chemistry Research

Laboratory, Mansfield Road, Oxford OX1

3TA, UK

Fax: +44 1865 285002

Tel: +44 1865 275961

E-mail: lorna.smith@chem.ox.ac.uk

E Paci, Institute of Molecular Biophysics,

School of Physics and Astronomy,

University of Leeds, Leeds LS2 9JT, UK

Tel: +44 113 3433806

E-mail: e.paci@leeds.ac.uk

(Received 25 May 2005, revised 12 July

2005, accepted 3 August 2005)

doi:10.1111/j.1742-4658.2005.04898.x

Retinol-binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin Under acidic con-ditions retinol-binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state Simulation con-formers, with a radius of gyration at least 1.1 A˚ greater than that of the native state, contain on average 37% b-sheet secondary structure In these conformers the central regions of the two orthogonal b-sheets that make

up the b-barrel in the native protein are highly persistent However, there are sizable fluctuations for residues in the outer regions of the b-sheets, and large variations in side chain packing even in the protein core Signifi-cant conformational changes are seen in the simulation conformers for resi-dues 85–104 (b-strands E and F and the E-F loop) These changes give an opening of the retinol-binding site Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol-binding protein and transthyretin dissociates, and retinol is released at the cell surface

Abbreviations

ANS, 8-anilino-1-napthalenesulphonate; MD, molecular dynamics; RBP, retinol-binding protein; Rg, radius of gyration; RMSD, root-mean-square deviation; TTR, transthyretin.

restraints in a novel approach to determine the free

energy landscape of this molten globule [9] In the

majority of the proteins whose molten globule states

have been characterized, both by experimental and

the-oretical methods, predominantly a-helical secondary

structure persists in the molten globule state [10–13]

In contrast, in this paper we concentrate on a protein

which is rich in b-sheet secondary structure in the

native state, and which retains the majority of this

b-sheet secondary structure in the molten globule state,

human serum retinol-binding protein (RBP)

RBP is a member of the lipocalin superfamily [14]

The proteins in this superfamily adopt a similar fold;

an eight-stranded up-and-down b-barrel with a

C-ter-minal helix However, they have a wide range of

func-tions, and high levels of sequence divergence, with

many members sharing under 20% sequence identity

[14,15] The lipocalins are widely distributed

through-out the eukaryotic and prokaryotic kingdoms [16,17]

Many of the lipocalins act as transporters for small

nonpolar ligands, such as retinoids, haem,

phero-mones, lipids, prostaglandins and pigments [18] RBP

transports all-trans-retinol (vitamin A) from its storage

sites in the liver to target tissues [19] It is postulated

that the local decrease in pH at the surface membrane

of target cells triggers the release of retinol, by a

mech-anism that is dependent on the conversion of RBP to

a molten globule state [20,21] It is possible that similar

mechanisms may prompt ligand release for other

mem-bers of the lipocalin superfamily For example, the

release of lipid ligands by human tear lipocalins under

acidic conditions is thought to be associated with a

transition to the molten globule state [22,23]

The molten globule state of RBP, formed under acidic

conditions, has been shown to exhibit the key

character-istics typical of these partially folded states Stoke’s

radii, from diffusion coefficient measurements, have

demonstrated that the molten globule retains a compact

fold [20] The mean molecular dimensions of the

parti-ally folded ensemble are only 13% larger than those

of the native state Far- and near-UV circular dichroism

(CD) spectra show that the protein contains a significant

level of secondary structure, but has a considerable level

of disorder in side chain packing, respectively [20,24,25]

Obtaining detailed information at an atomic level about

the molten globule state using techniques such as NMR

spectroscopy is challenging Partially folded states such

as molten globules are ensemble of interconverting

con-formers [26] Slow interconversion between populated

conformers gives rise to broadened NMR resonances,

while averaging of chemical shifts across the populated

ensemble gives a limited chemical shift dispersion [6,27]

Therefore, to develop a detailed model to further our

understanding of the molten globule state of RBP,

in vitroand in vivo experimental studies have been com-plemented with a molecular dynamics (MD) simulation study The results of this are reported here

It is not possible to explore adequately the conform-ational space accessible to partially folded proteins, within the simulation timescale currently accessible to conventional MD simulations of proteins in explicit solvent The exploration of non-native conformations

is therefore usually achieved, either by using very high temperatures in the simulations, or by introducing a suitable perturbation in a biased MD simulation, often using implicit solvent models (for a review of this topic see, e.g [28,29]) Explicitly modelling the perturbation induced by a change in the solution pH would not prompt the transition from the native to the molten globule state, on a timescale which can be directly simulated In this work therefore we have used three different perturbations in turn One perturbation forces

an increase in the protein radius of gyration, the sec-ond perturbation induces the breaking of native con-tacts in the structure and the third perturbation is aimed to speed up the exploration of diverse (in terms

of mutual RMSD) conformations These various per-turbations are applied using a particularly ‘soft’ time-dependent bias [30,31], designed to generate low energy pathways in the conformational space The large num-ber of diverse and moderately non-native conforma-tions generated with this biased molecular dynamics approach are then used as initial conformations for unperturbed, room temperature simulations This method allowed us to explore local free energy minima

in a broad region of the conformational space close to the native state The approach is designed to provide a qualitative map of the free energy landscape, in a region of the conformation space compatible with the experimental knowledge of the molten globule state

By applying this sampling approach to RBP, we are able to identify a broad basin of low energy partially folded conformers that are compatible with the avail-able experimental data [20,24,25,32–34] These con-formers provide a model for the molten globule state

of RBP that allows us to gain insight into the determi-nants of protein folding and the mechanism of retinol delivery and release, an important physiological prob-lem which remains unresolved

Results and Discussion

Sampling of conformational space

As described in the Experimental procedures section,

a biased MD simulation method has been used to

generate 160 diverse configurations of RBP Each of

these was then used as an initial configuration in an

unbiased MD simulation of 1.2-ns length Figure 1(A)

shows a plot of the heavy atom RMSD

(root-mean-square deviation) from the X-ray structure as a

func-tion of the radius of gyrafunc-tion (Rg), for structures taken

every 10 ps through the 160 unbiased MD simulations These data demonstrate the broadness of the conform-ational space explored in the study Analysis of the range of RMSD values seen for the MD conformers shows that there are three distinct peaks in the RMSD distribution (Fig 1B) These correspond to different levels of unfolding To aid the analysis, the conformers have been divided into three groups on the basis of their RMSD values Conformers in group 1 have an RMSD less than 4 A˚ and an Rg less than 16.5 A˚ Group 2 conformers have an RMSD in the range 4–7 A˚ and an Rg in the range 16.5–17.7 A˚, while group 3 conformers have an RMSD above 7 A˚ and an

Rg greater than 17 A˚ The characteristics of the group

1 conformers are very native-like, in keeping with their low RMSD values (< 4 A˚) For example, 81 of the residues that are in regions of secondary structure in the native protein have secondary structure popula-tions greater than 0.8 in the group 1 ensemble of con-formers We therefore focus our attention on the conformers in groups 2 and 3 that display a greater level of unfolding The native state structure of RBP has an Rg of 15.9 A˚ The 13% increase in molecular dimensions seen experimentally on forming the molten globule state would correspond to an effective Rg of

18 A˚ for the molten globule ensemble The group 3 conformers, with an Rgin the range 17–20 A˚, show an appropriate level of expansion on average for the mol-ten globule state (Fig 1A) The properties of this group of conformers have therefore particularly been compared with experimental data for this state

Secondary structure persistence The b-barrel in the native structure of RBP consists of eight b-strands (A-H) [35] These are arranged in two orthogonal b-sheets, with some of the b-strands being involved in both of the sheets The first sheet consists

of strands ABCDEF, and the second sheet contains strands EFGHA (Fig 2) The native structure also contains an a-helix (residues 146–158), which packs onto the b-sheet formed by the second set of strands All of the b-strands present in native RBP show a high level of persistence in the group 2 conformers (Figs 2 and 3) However, in many of the structures some of these strands are reduced in length, or have irregularit-ies compared to those in the native state For example, for strand F (residues 100–109) the mean b-sheet populations for the first five residues are only 0.05– 0.15 For strand E (residues 85–92) the mean b-sheet populations for the terminal residues 85 and 91–92 are 0.05–0.13, while those for residues 86–90 are 0.71–0.84 The disruption of this b-strand results predominantly

Å

0

0.1

0.2

0.3

0.4

0.5

RMSD

Rg

B

Rg (Å)

0

2

4

6

8

10

12

Group2 Group3

Group1

A

Fig 1 (A) Relationship between the protein radius of gyration and

the RMSD value from the X-ray structure of native RBP, for the

16 000 conformers taken at 10-ps intervals along the unbiased

simulations The radius of gyration and RMSD values are calculated

using all heavy atoms The black diamond corresponds to the native

state following the 2 ns equilibration simulation The definitions of

the three groups of conformers used in the analysis are shown (B)

The distribution of radius of gyration (filled bars) and RMSD values

(open bars) across the 16 000 simulation conformers.

from the loss of hydrogen bonds between strands E

and F, although in some structures the hydrogen

bonds between strand D and E are also missing The

a-helix also shows a high level of persistence in the

group 2 conformers, with a-helical populations in

the range 0.59–0.99 for the residues involved

In the group 3 conformers the changes are more

pronounced (Figs 2 and 3, and Fig 1 in the

supple-mentary material) All the b-strands now show

reduc-tions in length compared to those in the native

structure Even strand H (residues 129–138), one of

the particularly persistent strands, is reduced in length

by three residues in more than half of the structures,

with residues 129 and 130 having b-sheet populations

of less than 0.1 In addition, in group 3 strand E and

the first half of strand F are almost completely lost

Residues 85–92 and 100–104 show b-sheet populations

of less than 0.3 A significant disorder is seen across

the ensemble of group 3 conformers in the region

taining strand E, the first part of strand F and the

con-necting E–F loop (residues 85–104) The changes in

this region correlate with results from crystallographic

studies of bovine RBP These report a conformational

change in the E–F loop at low pH [34] In addition,

changes in this region were reported in a simulation of the apo form of RBP reported previously [36]

In almost all the group 3 conformers, however, a central region of the b-sheets is preserved Residues in the central regions of strands B, C, D, G and H and part of strand A have b-strand populations greater than 0.9 A persistent section comprising the central regions of strands B, C and D together with part of strand A in b-sheet 1, and the C-terminal region of strand F with the central parts of strands G and H in b-sheet 2, have residues with b-strand populations greater than 0.8 (Figs 2 and 3) Hence in the group 3 conformers the central area of each of the two ortho-gonal b-sheets, that make up the b-barrel in the native protein, are retained This is interesting as the two parts of the polypeptide chain that form these persist-ent cpersist-entral regions of the b-sheets have closely similar amino acid sequences In particular, the sequence of RBP contains an internal repeat with residues 36–83 (includes b-strands BCD) and 96–141 (includes b-strands FGH) having 34% identity [35] This may account, at least in part, for the similar behaviour of these regions in the simulations The central region

of the a-helix is also very persistent in the group 3

A B C D E

H

C

Fig 2 (A and B) The X-ray structure of human serum retinol-binding protein [35] (A) The b-strands are labelled, those in b-sheet 1 are shown in red and those in b-sheet 2 are shown in cyan The a-helix is blue and the retinol is magenta (B) Only the residues that have a sec-ondary structure persistence greater than 0.8 in the group 3 conformers (Fig 3) are coloured The figure was generated using the program MOLSCRIPT [53] (C) Backbone trace of representative structures from the three groups of simulation conformers (left, group 1; centre, group 2; right, group 3) In each case the average structure over the cluster centres is shown in red.

conformers, residues 151–156 having a-helical

popula-tions greater than 0.9

Experimental estimates of secondary structure

con-tent from far-UV CD spectra give 45 and 40% b-sheet

for the native and molten globule states of RBP,

respectively [26] The b-sheet estimate for the

native-state is in close accord with that observed in the X-ray

structure (46%) [35] In the group 3 simulation

con-formers, 53 residues have a b-sheet population of 0.60

or greater, and 11 residues have a b-sheet population

in the range 0.40–0.60 Taken together this

corres-ponds to 37% of residues in b-strand secondary

struc-ture, a value similar to that seen experimentally for the

molten globule state The experimental CD data show

that there is an increase in a-helical secondary

struc-ture on forming the molten globule state (8% native;

24% molten globule [25]) A large increase in a-helical

secondary structure is not observed, on average, in the

simulation conformers This difference may reflect

sampling and force field limitations in the simulations,

and the difficulty of interpreting experimental CD data

in a quantitative fashion The difference may also

reflect the fact that we do not model explicitly the con-ditions under which the molten globule is stable in the simulations, but rather identify conformers that are low in energy under native conditions However, although there is not a large increase in a-helical sec-ondary structure, the native state a-helix for residues 146–158 is essentially retained in all the simulation conformers In addition, in some of the conformers, particularly those in group 2, turns, some of a helical character, do form for residues 93–96 These residues are in the loop connecting strands E and F in the native protein, a region where the native structure is significantly disrupted in the simulations It is therefore possible that this is the part of the RBP sequence that forms non-native helical secondary structure when the molten globule state is adopted

Variations in side chain packing Despite the high persistence of the central regions of b-sheet secondary structure even in the conformers

in group 3, significant changes are observed in the

0

0.2

0.4

0.6

0.8

residue number 0

0.2

0.4

0.6

0.8

Fig 3 Fraction of the simulation conformers belonging to groups 2 (upper panel) and 3 (lower panel) in which certain secondary structure elements are present Secondary structure was calculated using the program DSSPcont [54] which identifies regions of secondary structure through an analysis of hydrogen bonding patterns b-sheet secondary structure is shown with open bars, helical (a, 310and p) secondary structure is shown with filled black bars, and turns and bends are shown with grey bars The secondary structure present in the native state

of RBP is indicated at the top of the figure, with the b-strands labelled A–H (open bars, b-strands; filled bars, a-helices).

packing of hydrophobic side chains The distances

between pairs of aromatic side chains and between

aromatic and other hydrophobic side chains, that are

close in the native state, have been analyzed in the

simulation conformers Some representative examples

of the distance distributions for conformers in groups

2 and 3 are shown in Fig 4 A very broad distribution

of distances across the simulation conformers is seen

when one of the side chains involved is from residues

85–104 This is the region that forms strand E and

part of strand F in the native structure, and is

disor-dered in many of the simulation conformers For

example, the distance Tyr90–Met73 ranges from 3.3 to

46.6 A˚, while the distance Phe36–Tyr90 varies from

5.9 to 52.0 A˚ in the group 3 conformers (Fig 4)

Fluctuations in the distances are seen even for

resi-dues in regions where the native structure is retained

to a significant extent Here though, the variations are

more limited Thus for Phe20–Phe137 and for Phe36–

Tyr133 the distance ranges are 3.2–15.3 A˚ and

2.9–13.8 A˚, respectively, in the group 3 conformers

(Fig 4) These changes in the packing of aromatic side

chains in the group 3 conformers are in accord with

the experimental loss of a near-UV CD spectrum on forming the molten globule state of RBP [20,24,25] These experimental data reflect the absence of fixed asymmetric environments for aromatic and cysteine residues in the molten globule

It is interesting that relatively short distances are retained for some of the side chains that are in contact with Trp24 in the native structure Thus the distances Trp24–Phe137 and Trp24–Arg139 are 3.0–6.8 A˚ and 2.7–8.4 A˚, respectively, in the group 3 conformers (Fig 4) Mutational studies have shown that Trp24, and its side chain interactions, play an important role

in stabilizing the RBP structure, and potentially in pre-venting misfolding [25] Trp24 and Phe137 are in a hydrophobic cluster in the native structure, while the side chains of Trp24 and Arg139 form an amine– aromatic interaction that closes the base of the b-barrel structure These residues are part of an evolutionary conserved set of residues in the lipocalin superfamily, which it is suggested may fold on a faster timescale than nonconserved regions [15] In accord with this, stopped flow fluorescence studies have shown that Trp24 is in a near native-like hydrophobic environment

Fig 4 Normalized distributions of the distances between selected residues for the simulation conformers in groups 2 (top panels) and 3 (bottom panels) The shortest distances between atoms in these side chains in the native state structure of RBP are indicated in paren-theses (A) Met73–Tyr90 (native state 3.8 A ˚ ); (B) Phe36–Tyr90 (native state 6.9 A˚); (C) Phe20–Phe137 (native state 5.0 A˚); (D) Phe36–Tyr133 (native state 3.4 A ˚ ); (E) Trp24–Phe137 (native 3.2 A˚); (F) Trp24–Arg139 (native state 3.7 A˚).The scale on the y-axis is arbitrary but the same for each pair of residues.

during the early stages of folding [15] The

persist-ence of native-like contacts for Trp24 in the

simula-tion group 3 conformers suggests that these contacts

may be retained in the molten globule state This would

be consistent with the significance of this side chain,

and its contacts, for the folding and stability of the

protein

Common interactions may be topological

constraints in the molten globule

The simulation conformers in groups 1–3 all share the

same topology, while varying to a large degree in

sec-ondary structure persistence and side chain disorder

This study provides an ideal model system to

investi-gate whether there is a common set of native-like

inter-actions maintained within low energy partly unfolded

structures, which provide a putative model for the

molten globule For this purpose, we computed the

pairwise effective energy between pairs of residues,

averaged over all the structures belonging to the

differ-ent groups defined above A network of the

interac-tions was built, by linking together residues whose

pairwise interaction is larger than a threshold (2.5 kcalÆ

mol)1), and removing isolated residues The resulting

network is shown in Fig 5 for the very native-like

group 1 and for the molten globule-like group 3 struc-tures The networks were analyzed using a network principle termed ‘betweenness-centrality’ [37,38] This measure allows the identification of key nodes govern-ing the network of interactions in proteins [39,40] The light grey residues in Fig 5 have the highest between-ness score, and are therefore the most influential to the network The interaction network representing group 3

is simpler, and the set of key nodes is smaller than for group 1

Interestingly, the structure of the networks of the group 1 and group 3 conformers is similar, and the set

of residues that have a high betweenness in group 3 have also high betweenness in group 1 conformers These are Arg10, Lys12, Asn14, Val107, Glu108, Thr109, Tyr111, Val116, Arg139 and Arg155 All these

10 residues are clustered together on the second face of the

1 b-barrel, in the same overall region of the tertiary structure However, they reside in six different struc-tural elements, being located in the N-terminal region, on

2 b-strands F, G, H, in the F–G loop and in the C-terminal helix in the native RBP structure There are wide variations in the characteristics of the contacts between these residues For example, in some cases there are interactions between charged or polar side chain groups (e.g Glu108 has contacts to Lys12 and

R2 F45

S46

V42

A43 L35

A26 I41

E44 E16

G22 A130

S134 F135

E131

C174

A71

S21 R19

T128

A28

T56

R62

A55 A57

M53

S54

E33

G51

E39

G92

L37

S7

V6 V61

G59

L125

E49

L122

K85 M73

Y114 E72

E102 V74

G75

C4 E103

N124 W91

V93

K99

G100

W105 F86

T76

A84

T80

Y173

Q149 F77

C120

F137

L144 R166 R163

I106

Y165

A115

V69

E158

R153 K58

K17

Q156

Y118 K30 S132

K89

M88

T78

N101 K87

Q98

Q117

E68

K29

N40 N171

W24 V136

G127

Y90

H104

S119

Q38

R121

Y133

R60

M27 I168

R5

Y25 S138 T113

R10

R155 E140

E112

V107 V116 E108 K12 T109 N14

R139 Y111

R2 Q98

E102 N101

I106

C4

A115

Y114 Y90

K85

N124

V69

M73

A71

G75

E82

F77

S119

L125

G59

E158 N171

Q164

G172

Q149

Y173

R153

L144 T128

C129

F135

S134

V136

E140

F137 N66

C174

M27 E16

K17

E13

F36

S7

R19

E33 V6

E31

Y25

W24

S21

G22 Q38

E39 M53

S54

A55 A57

S46

F45 E44

V42

A43

T56

K58

K87

E68 V61 N40

K29

R60

R121 Y165

Q117

Y133

R166 K30

W105

S138 G127

E131

Y118

L122 A130 R163 S132

T113 R5

R139 R10

Y111 R155

V107 N14 V116 E108 T109 K12

Fig 5 Network of effective interactions averaged over the structures of group 1 (A) and group 3 (B) The average effective energy between pairs of residues including solvation free energy was computed as described in Paci et al [55] The network was built connecting pairs of residues, whose absolute value of the interaction is larger than 2.5 kcalÆmol)1, and that are more than four residues apart in the sequence The threshold of 2.5 kcalÆmol)1is somewhat arbitrary and is chosen so that the representation of the network of interactions is shown most clearly Residues represented as light grey ovals are those with a betweenness larger than 5% (graphical representation and betweenness obtained with the program VISONE [56]).

Arg155) while in other cases there are interactions

between pairs of hydrophobic groups (e.g Val107 has

contacts to Val116) In addition, six of these 10

dues are also among the most highly conserved

resi-dues and interactions in the lipocalin superfamily

(N14, V107, E108, T109, Y111, R139) [15] The

simi-larity of the results for the group 1 and group 3

con-formers suggests that many, but not all, of the

interactions that are crucial for determining the native

state fold of the protein are retained in the RBP

mol-ten globule Furthermore, the conservation of the

resi-dues with the high betweenness in RBP across the

lipocalin superfamily indicates the important role that

folding to a specific native state plays in determining

evolutionary selection The results for RBP are

partic-ularly interesting in the light of a recently reported and

complementary analysis of networks of conserved

interactions in the intracellular lipid-binding protein

family [41] Proteins in this family are similar to the

lipocalins having a b-barrel structure However, the

proteins are in general smaller than the lipocalins and

their fold is different, consisting of a 10-stranded

b-barrel with a helix-turn-helix motif near the

N-termi-nus From this work on the intracellular lipid-binding

protein family it was suggested that a network of

con-served hydrophobic side chain interactions in the

b-sheet region of the protein could provide a potential

folding nucleation site This has interesting parallels

with an earlier reported study of the lipocalins [15]

Retinol binding site

Retinol binds to RBP in the core of the b-barrel

When bound, retinol is almost totally encapsulated by

the protein, the b-ionone ring lying in the centre of the

protein with the isoprene chromophore stretching

along the barrel axis [35] RBP releases retinol under

low pH conditions, undergoing the transition to the

molten globule state The changes to the structure seen

in the group 3 conformers, especially the disruption in

the strand E to F region, lead to a significant opening

of the retinol binding site to solvent, and so would

prompt loss of bound retinol in the molten globule

state The simulation analysis therefore proposes that

changes to the E to F region play an important role in

allowing retinol release from RBP This proposal is

supported by data from studies of ligand binding to a

number of b-lactoglobulins, other members of the

lipocalin superfamily [42,43] Here, an opening and

closing of the E-F loop region has been shown to

pro-vide a mechanism for the binding and release of

lig-ands In the b-lactoglobulins the changes are localized

in the E–F loop, and are prompted by the protonation

of a glutamate side chain within the loop In RBP, the simulation results suggest that the disorder in the E to

F region is part of much more wide spread conforma-tional changes, as the protein undergoes the transition into the molten globule state

Prior to the simulations reported here, retinol was removed from the X-ray structure coordinates This gave an increase of 251 A˚2 in the solvent accessible surface area of the protein Comparisons of the side chain solvent accessibility of the X-ray structure of RBP (without retinol bound) with that of the group 3 conformers show that, on average, there is a further increase in mean accessibility of 6.8 A˚2 per residue for the group 3 conformers Many of the side chains that make contacts with retinol when it is bound to the native state have, however, much larger increases in mean side chain accessibility in the group 3 conform-ers For example, for Leu63, Met73 and Phe77 the mean side chain accessibility increases by 59.3, 85.6 and 77.9 A˚2, respectively (Fig 6) Experimentally the molten globule state of RBP has been shown to bind strongly the hydrophobic dye 8-anilino-1-napthalene-sulphonate (ANS), giving an intense fluorescence spec-trum [24] This property, characteristic of molten globules, is generally recognized to reflect the presence

of exposed hydrophobic surfaces The simulation group 3 conformers show that in the case of RBP the exposed hydrophobic side chain groups from the reti-nol binding site are likely to be responsible, at least in part, for the observed ANS binding

Transthyretin binding site RBP circulates in the blood stream in a one-to-one complex with a second serum protein, transthyretin (TTR) [32] This large complex prevents the smaller RBP from being filtered through the renal glomeruli [19] Upon reaching target tissues, RBP and trans-thyretin dissociate, and retinol is released for delivery

to the cells, in a yet undetermined mechanism Four regions of the sequence of RBP at the opening of the barrel make contacts with TTR [32] The residues in RBP in three of the regions involved in these contacts are identified in Fig 6 The protein C-terminus is the fourth region of RBP which makes contacts with TTR These residues were missing from the X-ray structure of RBP [35], and so were excluded from the simulations reported here The binding of TTR to RBP has been shown to stabilize the binding of retinol

to RBP Studies of the interactions of chimaera

of RBP and epididymal retinoic acid binding pro-tein (ERABP) with TTR have identified the region

of the RBP sequence that is key to providing this

stabilization [33] The residues concerned are those in

the E-F loop, where the most major changes in

struc-ture are observed in the group 3 simulation

conform-ers This suggests that the dissociation of the RBP–

TTR complex may be linked to release of retinol

bound to RBP, via conformational changes in the E–F

region of the protein In this respect it is particularly

interesting that, in some of the simulation conformers

analyzed, residues in the E-F region adopted helical

turns It is therefore possible that a conversion in

sec-ondary structure could be involved in the RBP–TTR

dissociation mechanism Further work is needed,

how-ever, to investigate this mechanism in more detail,

including a study of the role of the C-terminal region

of the RBP sequence which was excluded from the

simulations reported here

Comparison with other molten globule states

Overall, analysis of the partially folded conformers

generated for RBP, and comparison with experimental

data, indicates that the structures in group 3 can

pro-vide a model for the ensemble of conformers

popu-lated in the molten globule state of the protein These

conformers retain considerable secondary structure,

but show disorder in side chain packing, and have

exposed hydrophobic groups In the group 3

conform-ational ensemble the central strands of the two ortho-gonal b-sheets show a high persistence The structure

in the outer strands is more fluctuating in nature It is interesting to compare these secondary structure char-acteristics with those seen for the molten globule states

of other proteins that have been studied in detail experimentally Many of the proteins whose molten globules have been characterized have predominantly a-helical secondary structure in their native state, and this is largely retained in the molten globule state Moreover for proteins that contain both a-helices and b-strands in the native state, it is the a-helical part of the structure that particularly persists in the molten globule The b-sheet regions are more unstructured, in contrast to the results for RBP This is seen, for exam-ple, in human a-lactalbumin [12] and Escherichia coli ribonuclease HI [13] Comparison with data for the molten globule state of two proteins which have a native state fold that is rich in b-sheet secondary struc-ture, carbonic anhydrase and b-lactoglobulin, is there-fore particularly relevant

Human carbonic anhydrase II contains 10 b-strands

in the native state, but differs topologically from RBP, having an ab roll fold [44] In the partially folded state the central strands 3–7 have native-like structure, but less ordered structure is found in the peripheral strands [45,46] Hence, as in the case of RBP, the core part of

-100

-50

0

50

residue number

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-50

0

50

Group2

Group3

Fig 6 Difference between the average side chain accessibility of each residue in the group 2 (upper panel) or group 3 (lower panel) simula-tion conformers and the side chain accessibility in the X-ray structure of native RBP (with retinol removed) Black bars indicate the residues that make contacts to bound retinol in the native state, and triangles show the residues that are involved in binding to TTR The solvent accessible surface area of the side chains has been calculated using the program NACCESS [57] The average increase in surface accessible area per residues is 6 and 7 A˚2 in the structures belonging to group 2 and group 3, respectively, compared to a change of )0.5 A˚ 2 in the equilibration (control) simulation.

the b-sheet secondary structure is retained in the

mol-ten globule state, while external regions of the

struc-ture are more disordered b-lactoglobulin is a member

of the lipocalin superfamily, and like RBP its native

structure contains an up-and-down b-barrel and one

C-terminal a-helix [42] Hydrogen exchange studies of

the molten globule state of equine b-lactoglobulin

show that the high protection factors are seen for

dues in strands G and H (values > 100 for five

resi-dues) These strands are linked by a disulphide bridge

between Cys106 and Cys119 Moderate protection

fac-tors (10–20) are seen for some residues in the regions

corresponding to strands A, D and F in the native

state, and also the a-helix Residues out of these

regions have protection factors less than 10 (with the

exception of Asn53 in the B–C loop which has a

pro-tection factor of 11) [47] These data are consistent

with a high persistence for native-like secondary

struc-ture in the central parts of the second b-sheet (residues

in strands F, G and H together with part of strand A),

while some secondary structure of a less persistent

nat-ure in the centre of the first b-sheet (particularly for

strand D) and in the a-helix [47] Results for bovine

b-lactoglobulin are very similar, with a high level of

hydrogen exchange protection being seen in the central

parts of the second b-sheet (residues in strands F, G,

H and A) in the partially folded state formed at pH 2

[48] In addition, for bovine b-lactoglobulin significant

hydrogen exchange protection persists for residues in

b-strands G and H in the cold denatured state [49]

These data for b-lactoglobulin are similar to the results

reported here, although in the RBP simulation

con-formers there is an equivalent level of persistence in

the central regions of both of the two b-sheets, while

in b-lactoglobulin the second sheet predominates The

proposed similarity between the molten globule states

of RBP and b-lactoglobulin suggests that the partially

folded protein characteristics identified here may be

typical of those adopted by other proteins in the

lipo-calin superfamily

Conclusions

In the model for the molten globule state of RBP

reported here the protein retains a persistent b-sheet

core, although even in these regions there is

consider-able variation in the side chain packing Out of the

b-sheet core there is much more disorder across the

conformational ensemble The network analysis of

the simulation conformers shows that there is a small

subset of persistent key interactions The most

signifi-cant changes in the structure are seen in the region

extending from the start of b-strand E to the middle of

b-strand F (residues 85–104) It is possible that the conformational changes in this region of the protein in the simulation conformers, including the presence of some a-helical character, could play an important role

in the mechanism for the dissociation of the TTR– RBP complex and retinol release at the cell surface Moreover, similarities between the simulation con-formers of RBP and experimental data for b-lactoglob-ulin, suggest that the model for the molten globule of RBP reported here may have relevance to our under-standing of the properties and ligand binding of other members of the lipocalin superfamily This is of partic-ular interest in the light of the development of engineered lipocalins for carrying novel ligands in therapeutic approaches [23,50,51]

Experimental procedures

The initial structure used in the simulations was the crystal structure of human serum retinol-binding protein at 2 A˚ resolution (PDB entry 1RBP [35], Fig 2), with the bound retinol ligand removed The C-terminal region of the RBP sequence is missing from the X-ray structure, as these resi-dues could not be located in the electron density map (resi-dues 175–182) We therefore also chose to exclude these eight C-terminal residues from the simulations reported here, although it is possible that this truncation of the sequence could affect the stability of non-native states of the protein An equilibration (control) simulation was run for 2 ns at 300 K The exploration of the conformational space outside the native state at room temperature was car-ried out by a biased molecular dynamics (BMD) scheme, similar to that used by Paci et al [30] All simulations were performed in implicit solvent (EEF1 [52]) The system was initially energy minimized to remove bad contacts, heated

up to 300 K in 0.6 ns and then equilibrated for 2 ns Dur-ing the last 1 ns of this equilibration simulation the average RMSD from the native state crystal structure was 2.4 (3.2) A˚ for Ca (all atoms): i.e the native state is relat-ively stable with the implicit solvation method employed Following this, a 6 ns simulation was performed using biased molecular dynamics, with a perturbation increasing the protein radius of gyration During this simulation, the radius of gyration and solvent accessible surface area of the protein increased up to 30% relative to that of the native structure, while the heavy atom RMSD from the native structure reached 13 A˚

Five conformations were picked out from along the tra-jectory, at approximately equal intervals in radius of gyra-tion value, ranging from one with an almost native-like radius of gyration to a maximally denatured conformer Five 1.6-ns simulations, one for each initial configuration, were started with a perturbation favouring the loss of native contacts between side-chain heavy atoms A configuration