Báo cáo khoa học: Preparation and characterization of geodin A bc-crystallin-type protein from a sponge pot

Here we report the preparation of geodin as a recombinant protein from Escherichia coli, its characterization through physico-chemical analyses, and a model of its 3D structure based on

A bc-crystallin-type protein from a sponge

Concetta Giancola1, Elio Pizzo2, Antimo Di Maro3, Maria Vittoria Cubellis2and Giuseppe D’Alessio2

1 Department of Chemistry, University ‘Federico II’ of Naples, Italy

2 Department of Biological Chemistry, University ‘Federico II’ of Naples, Italy

3 Department of Life Sciences, Second University of Naples, Italy

Vertebrate crystallins are proteins that last an entire

life-time, tightly packed in the eye lens which they provide

with the appropriate refractive index essential for vision

There are three families of crystallins: the a-crystallins,

complex multimers made up of small proteins which

perform also chaperon-like functions; and the b- and

c-crystallins, which together constitute a superfamily of

homologous proteins including monomeric c-type and

oligomeric b-type proteins [1] bc-Crystallins have been

extensively studied as models of molecular evolution

[2–5] and for their structural features [6,7] The 3D

structures of many members of the superfamily have

been determined by X-ray crystallography [8–11], and

NMR [12,13] They indicate that the smallest structural

unit in the bc-crystallin superfamily is a b-stranded

Greek key motif, with two such motifs making up a

domain; two domains connected by a peptide linker

constitute a c-crystallin-type monomer, or a subunit of

a b-crystallin-type oligomer

A long evolutionary history can be traced for bc-crystallins, back to single-domain homologues from moulds [14], and bacteria [12], and a two-domain homologue from amphibians [15] Recently, a bc-crys-tallin-type gene from a sponge, Geodia cydonium, has been identified [16] and cloned [17] The finding that this is an intron-less gene [17] (as compared with ver-tebrate bc-crystallin genes, all of which are endowed with several introns) and the very early divergence of porifera ) the most primitive metazoans ) support the idea that the protein encoded by this gene, called geo-din, is the most ancient member of the metazoan bc-crystallin-type superfamily Thus, it appeared to be

of interest to inspect the structural features of this pro-tein, to verify the hypothesis that the same structural

Keywords

geodin; bc-crystallins; metazoans;

calorimetry; protein stability

Correspondence

G D’Alessio, Department of Biochemistry,

University of Naples Federico II, Via

Mezzocannone 16, 80134 Naples, Italy

Fax: +39 081 5521217

Tel: +39 081 2534731

E-mail: dalessio@unina.it

(Received 4 November 2004, revised 30

November 2004, accepted 20 December

2004)

doi:10.1111/j.1742-4658.2004.04536.x

Geodin is a protein encoded by a sponge gene homologous to genes from the bc-crystallins superfamily The interest for this crystallin-type protein stems from the phylogenesis of porifera, commonly called sponges, the earliest divergence event in the history of metazoans Here we report the preparation of geodin as a recombinant protein from Escherichia coli, its characterization through physico-chemical analyses, and a model of its 3D structure based on homology modelling Geodin is a monomeric protein of about 18 kDa, with an all-beta structure, as all other crystallins in the superfamily, but more prone to unfold in the presence of chemical denatu-rants, when compared with other homologues from the superfamily Its thermal unfolding, studied by far- and near-CD, and by calorimetry, is des-cribed by a two-state model Geodin appears to be structurally similar in many respects to the bacterial protein S crystallin, with which it also shares

a significant, albeit more modest stabilizing effect exerted by calcium ions These results suggest that the crystallin-type structural scaffold, employed

in the evolution of bacteria and moulds, was successfully recruited very early in the evolution of metazoa

Abbreviations

DSC, differential scanning microcalorimetry; GuHCl, guanidine hydrochloride; TFA, trifluoroacetic acid.

scaffold was used in the evolution of bacteria and

moulds, and then very early recruited in the

metazo-ans Here we report the expression and purification of

recombinant geodin and its thermodynamic and

spect-roscopic characterization, and propose a putative

structure of the protein, as derived through homology

modelling

Results and Discussion

Preparation of recombinant geodin

As described in Experimental procedures, E coli cells

transformed with the cDNA encoding geodin, the

putative bc-crystallin-type protein from the sponge

G cydonium (UNIPROT accession number: O18426),

were lysed by sonication followed by centrifugation

By SDS⁄ PAGE, both the resulting supernatant and

pellet were found to contain a protein of
18 kDa,

expected for geodin on the basis of the amino acid

sequence encoded by the available gene sequence

(Fig 1)

Thus, both fractions were investigated: the soluble

fraction (S preparation) and the pelleted fraction, after

solubilization and renaturation (see Methods), called

IB preparation The S and IB preparations were

fract-ionated in parallel by size exclusion chromatography

followed by ion exchange chromatography The

chro-matography runs, described in the Experimental

proce-dures, are illustrated in Fig 2

When the proteins purified from the S or IB prep-arations, respectively, were analysed by SDS⁄ PAGE, they were both found to contain a single protein with the molecular size of geodin (Fig 1) Identical results were obtained when the two preparations

100 kDa

66 kDa

14 kDa

8 kDa

18 kDa

M 1 2 3 4

Fig 1 SDS ⁄ PAGE of protein preparations from the lysate of E coli

cells transformed with geodin encoding cDNA Lane 1, IB

prepar-ation from inclusion bodies; lane 2, S preparprepar-ation from the lysate

soluble fraction; lane 3, geodin purified from the IB preparation;

lane 4, geodin purified from the S preparation.

A

B

C

D

Fig 2 Gel filtration on Superdex G-75 of the proteins from the S preparation (A) and the IB preparation (B) from the lysate of E coli cells transformed with geodin encoding cDNA The inserts show the results of SDS ⁄ PAGE separations of individual fractions from each column as indicated by fraction numbers Fractions 48 through 56 were pooled and dialysed Each pool was then chroma-tographed on a cation exchange column (Resource-S) as illustrated

in C (for the S preparation) and D (for the IB preparation).

were subjected to RP-HPLC on a C4 column as

des-cribed in Methods Also by this procedure, the

pro-tein, isolated from the S or IB preparation, was

found to be homogeneous and eluted in a single,

symmetrical peak from the HPLC column (data not

shown)

The two proteins eluted from the HPLC column,

analysed by MALDI-TOF MS, were found to have

the following molecular masses: 17 788, protein

puri-fied from S preparation; 17 785 protein puripuri-fied from

IB preparation These values compare very

satisfactor-ily with the geodin mass value calculated from its

amino acid sequence (17 781 kDa)

The amino acid sequence determination carried out

on the proteins purified from either the S or IB

prepa-rations gave the following N-terminal sequence

(one-letter code), identical for both protein preparations:

NH2–STAKVTLVTSGGSSQDFT–, which is exactly

the sequence deduced for the N terminus of the protein

encoded by the geod gene from G cydonium

These results indicated that: (a) the same protein

was contained in both the S and IB protein

prepara-tions, as derived from the soluble and insoluble

frac-tions, respectively, of the E coli lysate; (b) this protein

was geodin, the expression product of the geod gene

from the sponge G cydonium

Thus, a single form of geodin was expressed by E coli

under the conditions described above The finding that

the expressed recombinant geodin was distributed

between cytosol and inclusion bodies of transformed

E coli cells can be explained by considering that the

E coli synthetic machinery allowed the production of

free, soluble geodin up to a solubility limit, beyond

which the excess protein was sequestered in inclusion

bodies

Stability against urea and guanidinium chloride Figure 3 shows the CD spectra of geodin in far-UV and near-UV at T¼ 20
C and pH 5.0 and 7.0, respectively At both pH values, the far-UV CD spec-trum of the protein exhibits a strong positive band at

217 nm, which indicates a well defined b-conformation

in solution This suggests that geodin is an all-b pro-tein The different protonation state apparently does not affect the secondary structure, as no drastic chan-ges are observed in the far-UV signal (Fig 3) In the near-UV region the effects of protonation are signifi-cant in the protein tertiary structure with a higher exposure of chromophors at pH 5.0

As for vertebrate crystallins) such as mammalian

cB and bB2 crystallins) geodin tends to aggregate, especially at higher temperatures and concentrations However, because at relatively high concentrations geodin was more soluble at pH 5.0 than at pH 7.0, a

pH value closer to geodin pI value of 7.9, as calculated for from its amino acid sequence, we chose to study the thermodynamic properties of the protein at pH 5 Geodin stability against chemical denaturants was investigated by measuring the molar ellipticity at

217 nm, and the shift in fluorescence maximum wave-length, as a function of urea or guanidine (GuHCl) concentration As shown in Fig 4, all denaturation curves have monophasic, sigmoidal shapes with single midpoints of denaturation determined at 3.5 m and 1.2 m for urea- and GuHCl-induced denaturation, respectively When the denatured protein solutions were dialysed, their far-UV CD spectra were found to

be identical to that of the native protein This indicates that the unfolding of geodin as induced by chemical denaturants is reversible

Fig 3 Far-UV (A) and near-UV (B) CD

spec-tra of geodin at pH 5.0 (solid lines) and

pH 7.0 (dashed lines).

Geodin appears to be very sensitive to chemical

denaturation, in fact more sensitive than a typical

crystallin-type protein of the vertebrate c family

(Table 1) In particular, with guanidinium⁄ HCl as a

denaturant, the midpoint of denaturation determined

for geodin is lower than those determined for other

monomeric crystallin-type proteins, but very close to

that determined for spherulin 3a The latter

crystal-lin-type protein is a homodimeric protein in which

two single-domain protomers associate into the

topo-logical equivalent of a monomeric crystallin Thus,

the finding of a similar dependence from chemical

denaturation between geodin and spherulin 3a might

be suggestive of weaker inter-domain interactions in

geodin, weaker than those at the interfaces of monomeric crystallins, but comparable with those

in a noncovalent two-domain homologue such as spherulin 3a

As for the denaturation of geodin in urea, as com-pared with other monomeric crystallin-type proteins, our findings (Table 1) suggest that the stability of geo-din in urea resembles that of protein S rather than that

of a mammalian crystallin such as cS-crystallin, a qualitative, indirect indication of a closer structural relationship between geodin and protein S Geodin also displays a single denaturation midpoint, detectable

at pH 5, but with no aggregation at any urea concen-tration (Fig 4, Table 1)

Fig 4 (A, B) Urea and (C, D) GuHCl-induced transitions of geodin at pH 5.0 The transi-tions were monitored by the shift in the wavelength corresponding to the maximum

of the fluorescence spectrum (A and C), and

by far-UV CD at 217 nm (B and D).

Stability against temperature

When the thermal denaturation of geodin was

investi-gated by the dichroic absorption in far- and near-UV

(Fig 5), at 217 nm and 280 nm, respectively, the

curves showed cooperative transitions in agreement

with a two-state model For both, the midpoint

tem-perature was found to be 61
C, which indicates a

sim-ultaneous collapse of secondary and tertiary structures

The calorimetric profile obtained by differential

scan-ning microcalorimetry (Fig 6), was not influenced by

protein concentration, although the endothermic peak

was followed by an exothermic process that coincides

with aggregation and precipitation, which lead to an

irreversible overall denaturation process

A description of an irreversible protein denaturation

is in the model proposed by Lumry and Eyring [18]:

N$ U $ F where N is the native protein, U is the reversibly

unfolded protein and F is the final, irreversibly

dena-tured protein Starting from the Lumry–Heyring

model, Sanchez-Ruiz [19] has shown that when the

transition is calorimetrically irreversible, but the

irre-versible step takes place with a significant rate at a

temperature even slightly above those corresponding to

the transition, the equilibrium thermodynamic analysis

is permissible

In this case the unfolding process:

N !K U

is described by the van’t Hoff equation:

@ln K

@T

P

¼DH 0

where K is the equilibrium constant and DH0 is the enthalpy that determines the variation of K with the absolute temperature The integrated form can be writ-ten as:

K¼ exp DH

0ðTÞ R

 1

T

1

Tm

ð2Þ

At each temperature value, the enthalpy of this ther-modynamic system can be described as:

Table 1 Physico-chemical data for geodin and other two-domain

crystallin-type proteins Values of Tm, DH 0 and DG 0 , determined by

DSC, are from reference [39] for protein S, and reference [26] for

spherulin 3a and human-cS Data for denaturation with chemical

denaturants were obtained by CD spectroscopy for protein S [22],

spherulin 3a and human-cS [25].

c 1 ⁄ 2 GuCl

( M )

c 1⁄ 2 urea ( M )

T m (
C)

DH 0 (kJÆmol)1)

DG 0 (293 K) (kJÆmol)1) Geodin 1.2 a 3.5 a 61.0 c 532 41

1.2b 3.6b 60.5d

+ Ca 2+ 65.0 c,d 570 48

Human-cS 2.6 8.0 75 750 84

Protein S 1.7 3.7 52 ⁄ 64 e

399 ⁄ 263 29 ⁄ 16 + Ca 2+ 1.9 4.8 64 ⁄ 65 e 454 ⁄ 332 39 ⁄ 25

Spherulin 3a 1.1 53.3 523 81

+ Ca 2+ 2.5 68.7 1020 137

a Data from near-UV CD spectroscopy b Data from fluorescence

spectroscopy. cData from near- and far-UV CD spectroscopy.

d Data from DSC e Values for first ⁄ second transition.

Fig 5 Temperature-induced unfolding transitions at pH 5.0 monit-ored by far-UV CD (A) and near-UV CD (B).

HðTÞ ¼ fNHNþ fDHD¼ HNþ fDðHD HNÞ

where fN and fD are the fractions of molecules in the

native and denatured states, respectively HN and HD

are the corresponding enthalpies of native and

dena-tured states

Choosing the native state as reference state, the

fol-lowing equation for the excess enthalpy is obtained:

<DH0ðTÞ > ¼ HðTÞ  HN ¼ fDDH0ðTmÞ

¼ ½K=ð1 þ KÞ  DH0ðTmÞ ð4Þ which, derived with respect to the temperature and

based on Eqn (1), leads to:

<DC0ðTÞ > ¼½DH

0ðTmÞ2

RT2 ½K=ð1 þ KÞ2

þ DC0

PðTmÞ½K=ð1 þ KÞ ð5Þ This equation allows the simulation of a calorimetric

curve for a two-state transition [20,21] When the

experimental curve of geodin denaturation and the

DSC profile predicted by Eqn (5) were juxtaposed, a

satisfactory agreement was found (Fig 6) This

con-firms that the equilibrium thermodynamic treatment

can be applied to this case, and the Gibbs’ energy

value can be calculated

In Table 1 we compare the thermodynamic

parame-ters of geodin with those of two-domain crystallin-type

proteins for which values have been determined for

most physico-chemical parameters, such as monomeric

bacterial protein S and mammalian human-cS, and

spherulin, which is a two-domain noncovalent dimer

The latter two proteins, as well as geodin, display a single transition in the denaturation process Protein S instead undergoes a two-step denaturation, but the main transition is centred at 64.4
C, closer to that of geodin (61
C) when compared with the Tm values of

75
C and 53
C determined for human-cS and spheru-lin, respectively Furthermore, the values forDG
, as a parameter of thermodynamic stability, are of about

80 kJÆmol)1for human-cS and spherulin 3a, and of 41 and 45 kJÆmol)1for geodin and protein S, respectively The value of 45 kJÆmol)1for protein S is the value cal-culated for the overall unfolding process Thus, the conclusion can be proposed that, based on thermody-namic behaviour, geodin is a crystallin-type protein closer to protein S than to a mammalian (human-cS)

or mould (spherulin 3a) crystallin

Stability of geodin in the presence

of calcium ions Several two-domain members of the bc-crystallin superfamily are stabilized by calcium ions, such as pro-tein S [22], spherulin 3a [14,23,24], and c-crystallin [25] To investigate the effect of Ca2+on geodin stabil-ity, DSC and CD measurements were performed in parallel at pH 5.0 in ammonium acetate buffers con-taining either 1 mm CaCl2 or 1 mm Na2EDTA The near-UV spectrum, shown in Fig 7A, indicates that upon CaCl2 addition the positive band at 280 nm increases, revealing perturbation in the exposure of some aromatic residue(s)

Also the far-UV dichroic spectrum is affected, as shown in Fig 7B, with a less intense minimum at

217 nm and a shoulder at about 230 nm in the pres-ence of Ca2+ The latter findings, which suggest a reas-sessment in the secondary structural order of the protein upon calcium binding, are in contrast with the results obtained in the case of homologous protein S, for which calcium binding affects only the protein ter-tiary structure [26]

The CD melting profile determined for geodin at

217 nm in the presence of Ca2+ was found to be sig-moidal as that obtained in the absence of calcium ions, but the midpoint denaturation was increased to 65
C (Fig 7C) A perfectly coincident value was obtained

by calorimetric measurements in the presence of cal-cium ions (Table 1) The higher melting temperature (increased by 4–5
C) and higher denaturation enthalpy (Table 1) indicate that calcium ions provide

an additional stabilization to geodin In fact, the calcu-latedDG0at 293 K was found to be 48 kJÆmol)1, com-pared to 41 kJÆmol)1 calculated from measurements in the absence of Ca2+ions

Fig 6 Experimental (solid line) and simulated (dotted line)

calori-metric curves of geodin at pH 5.0.

A structural model for geodin

We first searched for homologues of geodin in

Hom-strad (http://www-cryst.bioc.cam.ac.uk/data/align), a

curated database [27] of structure-based alignments for

homologous protein families, which makes use of

FuGUE (http://www-cryst.bioc.cam.ac.uk/fugue/

prfsearch.html [28] The first hit found by fugue (z

score¼ 20, average sequence length ¼ 174)

correspon-ded to the family of bc-crystallins The second hit (z

score¼ 13.5, sequence length ¼ 101) corresponded to

spherulin 3a, a homodimer in which each chain

con-tains a single crystallin-type domain [24] The same

two hits with statistically significant z scores were

obtained when the sequence of geodin was divided into

two halves and each half was used independently as a

query sequence Therefore, it is reasonable to assume

that geodin (sequence length¼ 163) has two tandemly

repeated domains, each with a crystallin-type fold We

submitted the geodin sequence to a threading server

(http://www.sbg.bio.ic.ac.k/~3dpssm), and obtained the

same fold

Geodin was then aligned with a structure-based

available alignment of bc-crystallins with

experiment-ally solved structures, stored in Homstrad and

decor-ated with joy [29] This makes visible structural

features, e.g buried residues (in upper case), exposed

residues (lower case) (Fig 8) The alignment of geodin

to this pre-existent alignment of crystallins was carried

out using fugue and was manually modified in the

N-terminal portion The improvement produced was

tested by comparative model validation using anolea [30,31] and prosaii [32]

Buried residues belonging to b-strands and facing the intradomain hydrophobic core of each domain, as well as other buried residues and positive-u glycine residues were found to be mostly conserved in geodin, which indicated the good quality of the alignment It should be noted that the sequence of protein S shows the insertion of a valine at position 50, where the sequences of the other homologues show a glycine The alternative to this insertion in the Homstrad align-ment would have required both an insertion plus a preceding gap In the alignment by fugue of geodin a glycine could align with the glycines of the other homologues, and only a gap was required

Recombinant geodin is monomeric, as are c-crystal-lins and protein S c-Crystalc-crystal-lins have a symmetric inter-domain interface made up by the second motifs

of both domains whereas protein S has a different interface, made up by the second motif of the first domain and the first motif of the second domain The inter-domain interface in c-crystallin (PDB code 4gcr)

is quite extended, made up of hydrophobic interactions between the triad M43⁄ F56 ⁄ I81 in the N-terminal domain and the other triad V132⁄ L145 ⁄ V170 in the C-terminal domain, and Q54–F145 and F56–Q143 hydrogen bonds These positions, marked by * in the alignment (shown in Fig 8), do not appear to be com-pletely conserved in geodin, as some of the hydro-phobic residues involved in c-crystallin are replaced by nonhydrophobic residues

Fig 7 The effects of Ca ions on geodin structure Near-UV (A) and far-UV (B) CD spectra of geodin at pH 5.0 in the presence of Na2EDTA (dotted line) or 1 m M CaCl 2 (solid line) In (C) the temperature unfolding transition of geodin in the presence of Ca2+(1 m M , solid line) is compared to that recorded in the absence of Ca 2+ (1 m M Na2EDTA, dotted line).

The inter-domain interface in protein S (PDB code

1 prs) is instead less extended, made up of the

V65⁄ A67 ⁄ Y121 triad, and a N68–D107 hydrogen bond

[13] These positions, marked in the alignment by #,

are not conserved in geodin

Therefore, neither alignment could explain how

domains are assembled in geodin To infer the relative

orientation of domains in geodin, two independent

models were built with modeller As templates we

used, respectively, c-crystallins (PDB codes 4gcr,

1elp,1a45,1a5d) for a ‘mod-gamma’, and protein S

(PDB code 1 prs) for a ‘mod-prs’ anolea [30,31] and prosaii [32] were run on the models and, as a control,

on the template structures Even if the energy calcula-ted for c-crystallins is much lower than that calculacalcula-ted for protein S, anolea indicated that the energy of the model based on 1 prs is comparable with the energy of the model based on c-crystallins, while prosaii indica-ted that the model based on 1 prs is much better than that based on c-crystallins

Figure 9 shows that in mod-prs, the model built with protein S as a template, the inter-domain

Fig 8 Alignment of geodin amino acid sequence with the sequences of homologous bc-crystallins The sequences of bovine (PDB codes 1elp, 1a45, 4gcr, 2bb2) and murine (1a5d) crystallins, and of spore coat protein S from Myxococcus xanthus (1 prs) are decorated as follows: blue for b-strands; red for a-helices, brown for 3–10 helices Buried residues are in uppercase letters; residues with a positive u angle, in ital-ics; hydrogen bonds to main-chain amides in bold; hydrogen bonds to main-chain carbonyls are underlined Buried residues belonging to b-strands and facing the intra-domain hydrophobic core of each domain, other buried residues, and positive-u glycine residues conserved in geodin, are highlighted in green, blue and yellow, respectively The inter-domain interface residues in 4gcr are marked by asterisks The inter-domain interface residues in 1 prs are marked by #.

interface is hydrophobic, as made up of W57, I58,

L101, P102 and P106 (shown as yellow as

ball-and-stick residues) Furthermore, correctly intercalated

resi-dues were found, both positively charged (K59 and

R107; blue ball-and-stick residues), and negatively

charged residues (D61 and D100; red ball-and-stick

residues, Fig 9) These charged residues, located at the

surface of the two domains, could also contribute with

hydrogen and⁄ or ionic bonds to the stability of geodin

inter-domain interface As these interactions were not

used as restraints with modeller, they further validate

the model built on protein S, and shown in Fig 9 On

the other hand, the model built using c-crystallins as

templates did not possess a hydrophobic interface or

any other interactions capable of stabilizing

mono-meric geodin

Since the key question at hand was to predict the

relative orientation of domains, we submitted the

mod-gamma, to a protein–protein interaction server,

http://www.biochem.ucl.ac.uk/bsm/PP/server [33] The

results, summarized in Table 2, indicated that the

interface in mod-prs is larger, more planar, although

less circular than that in mod-gamma The interacting

surfaces in mod-prs are more complementary, as

proved by a lower gap volume and gap volume index

(gap volume⁄ interface ASA), with a balanced number

of hydrophobic residues and, interestingly, a higher

number of interdomain hydrogen bonds, and salt

brid-ges In conclusion, the analysis confirms the results

reported above: although mod-prs and mod-gamma

represent alternative ways to assemble the two

domains of geodin, measurement of several descriptive

parameters of the interfaces and visual inspection both suggest that in geodin, as in protein S, the inter-domain interface is made up by the second motif in the first domain and the first motif of the second domain

As it has been found that geodin is stabilized by

Ca2+(see above), the 3D model of geodin with protein

S as a template was analysed to verify whether it could accommodate a Ca2+ binding site Protein S has two binding sites for Ca2+, one per domain They are formed by residues in the folded hairpin of the first Greek key motif and by residues in the loop connect-ing the penultimate and ultimate strands in the second Greek key motif of each domain The site with the highest affinity for Ca2+ in protein S is in the N-ter-minal domain and is defined by residues E10 and E71

It can be proposed that T10, S11 and E62, located in hydrophilic loops in the corresponding region of geo-din, could play the same role This tentative Ca2+ binding site was thus modelled (green ball-and-stick residues) in the geodin structure shown in Fig 9 Another presumable but weaker binding site could be identified in the C-terminal domain of geodin, defined

by Asn93 and Asn145

Spherulin 3A, a single-domain mould protein with crystallin fold, has two distinct Ca2+binding sites per domain, one site is located between the folded hairpin

of the first Greek key motif and the loop between the last two strands of the second Greek key motif [13] This site corresponds to the sites identified in protein S and modelled in geodin

Conclusions

The results of the physico-chemical studies reported above, and those from homology modelling, lead to

Fig 9 A model of geodin built using the structure of protein S

(1 prs) as a template Inter-domain interface residues are shown as

yellow ball-and-stick residues when they are hydrophobic; in red

when they are negatively charged; in blue when they are positively

charged Residues proposed to describe the binding site for Ca2+

are in green ball-and-stick notation The structure was drawn using

MOLSCRIPT [37] and rendered using RASTER 3 D [38].

Table 2 Domain–domain interface analysis of alternative models of geodin domain assembly.

Mod-prs domain-1

Mod-prs domain-2

Mod-gamma domain-1

Mod-gamma domain )2 Interface ASA 643.29 741.38 415.18 404.38

% Interface ASA 13.62 14.73 8.92 8.29 Planarity 2.14 2.13 1.02 1.28 Length ⁄ breadth 0.49 0.54 0.74 0.71

% Polar atoms

in interface

38.09 43.61 32.79 64.11

% Non–polar atoms in interface

61.90 56.30 67.20 35.80

Hydrogen bonds 3 3 1 1

Gap volume 1644.62 1644.62 2927.67 2927.67 Gap volume index 1.19 1.19 3.57 3.57

the first structural description of geodin, and validate

its identification as a homologue of the bc-crystallin

superfamily Geodin is a monomeric, two-domain

protein, made up of b strands apparently folded in

Greek-key motifs, just as its mammalian, amphibian

and bacterial homologues Of particular interest is that

its source is a sponge, G cydonium, which makes

geo-din the most primitive metazoan bc-crystallin studied

so far

By both fluorescence and CD spectroscopic analyses,

geodin is found to be, with respect to other

bc-crystal-lins, more readily but reversibly unfolded by chemical

denaturants, with a single midpoint of denaturation,

both in urea and GuHCl These features are very

sim-ilar to those found for protein S [22] Consistently, our

conclusion from homology modelling strongly suggests

that the closest structural homologue to geodin is

bac-terial protein S

Geodin thermal denaturation, studied both by

near-and far-UV CD near-and calorimetry, shows that geodin

unfolds with a typical cooperative transition in

agree-ment with a two-state model, with a simultaneous

col-lapse of secondary and tertiary structures This is

difficult to explain, given the larger structural

differ-ences in primary structure and 3D architecture

between the two geodin domains, as they result from

the proposed sequence alignment and model,

com-pared with the more similar sequence and architecture

of the two domains in the other monomeric crystallins

studied so far It should be considered that, besides the

structural differences among the proteins under

com-parison, different pH values were adopted in the

experiments

The similarity in structural properties between geodin

and protein S extends to the stabilization exerted by

calcium ions on both crystallin-type proteins, although

the removal of calcium affects in geodin both

secon-dary and tertiary structure, whereas for protein S only

the tertiary structure is affected Furthermore, the

effect of calcium on geodin stability is less conspicuous,

with an increase inDG0 of denaturation of 7 kJÆmol)1,

compared to that reported for protein S (19 kJÆmol)1)

This outcome is due mainly to the higherDH0

contribu-tion for protein S denaturacontribu-tion in the presence of

Ca2+, in turn apparently due to strengthened

inter-domain interactions

As from the evolutionary point of view, it is of

interest that the crystallin-type structural organization,

already evolved in bacteria and moulds, was readily

recruited for porifera, the earliest metazoans

Further-more, it is interesting that in the model as derived for

geodin the inter-domain interfaces appear to be

stabil-ized by hydrophobic interactions, but also by a

number of polar interactions, such as H-bonds and salt linkages These contacts can be interpreted, as it has been proposed for vertebrate bc-crystallins [5], as remi-niscent of the polar, solvent exposed surfaces in the putative single-domain ancestor(s) that evolved to associate into two-domain geodin

Experimental procedures

Cloning and expression of geodin Cloning into the pET22b(+) vector (Novagen, Madison,

WI, USA) of the DNA segment encoding geodin, a puta-tive bc-crystallin-type protein from G cydonium, was carried out as described previously [17] The plasmid was used to transform E coli strain BL21 (DE3) (from AMS Biotechnology) For over-expression of geodin, the bacterial cultures were grown at 37
C to D600¼ 1, then induced by addition of 0.1 m isopropyl-1-thio-d-galactopyranoside After overnight growth at room temperature, cells were pel-leted by centrifugation, and lysed by sonication An Ultra-sonic Ultra-sonicator (Heat System UltraUltra-sonic) was used at

20 kHz, with 30-s impulses, each followed by a 30-s rest period, for a total time of 15 min

A protein with the molecular size of geodin was detected

by SDS⁄ PAGE as a soluble protein in the lysate superna-tant (henceforth termed S preparation), but also in the insoluble material (Fig 1) Thus, the insoluble material, washed twice in 50 mm Tris⁄ HCl pH 8 containing 20 mm

Na2EDTA, and once in 100 mm Tris⁄ HCl pH 8 containing

1 mm Na2EDTA, was solubilized by denaturation with 6 m GuHCl in 25 mm sodium phosphate pH 7 (buffer P) Rena-turation followed, obtained through extensive dialysis against the same buffer This yielded another preparation containing a protein with the mobility of geodin, henceforth termed IB preparation, as presumably it consists of proteins sequestered in inclusion bodies

Both preparations S and IB were fractionated by gel fil-tration, carried out on a column of Superdex G-75 (Amer-sham Biosciences) equilibrated with buffer P containing 0.3 m sodium chloride Figure 2 illustrates that the fraction-ation of both S and IB preparfraction-ations yielded three main UV absorbing fractions Only in the middle peak from either S

or IB preparations SDS⁄ PAGE runs revealed the presence

of a protein band with the molecular size expected for geo-din The corresponding fractions (Fig 2A,B, insets) were pooled and dialysed against 50 mm ammonium acetate buf-fer pH 5

The Superdex G-75 fraction pools obtained from the S and IB preparations were each loaded onto a cation exchange Resource-S column (Amersham Biosciences) equilibrated in 50 mm ammonium acetate pH 5, and eluted with a 60-min linear gradient from 50 to 300 mm ammo-nium acetate As shown in Fig 2C most of the protein